Biological processes for the production of aryl sulfates

ABSTRACT

The present invention generally relates to the field of biotechnology as it applies to the production of aryl sulfates using polypeptides or recombinant cells comprising said polypeptides. More particularly, the present invention pertains to polypeptides having aryl sulfotransferase activity, recombinant host cells expressing same and processes for the production of aryl sulfates employing these polypeptides or recombinant host cells.

TECHNICAL FIELD OF THE INVENTION

The present invention generally relates to the field of biotechnology asit applies to the production of aryl sulfates using polypeptides orrecombinant cells comprising said polypeptides. More particularly, thepresent invention pertains to polypeptides having aryl sulfotransferaseactivity, recombinant host cells expressing same and processes for theproduction of aryl sulfates employing these polypeptides or recombinanthost cells.

BACKGROUND OF THE INVENTION

A range of phenolic compounds are of great interest to the biotechindustry since they are building blocks for polymeric compounds.Examples of such phenolic compounds include p-coumaric acid (pHCA) orother hydroxycinnamic acids which form the basis for many secondarymetabolites including flavonoids and stilbenes. However, many of thesephenolic compounds are toxic to producing organisms, and thus limit theproductivity during fermentation. Hence, there is a need for large scaleproduction processes, and especially for biological large scaleproduction processes allowing improved productivity.

Moreover, a range of phenolic compounds, and especially those used asdrugs or food additives such as resveratrol or vanillin, show poorsolubility in water which makes it difficult for these compounds to beuptaken by the body. Hence, there is also a need for providing suchphenolic compounds in a form which improves the solubility, and hencebioavailablility, preferably by using biological large scale productionprocesses.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a method for largescale production of aryl sulfates. Furthermore, it is an object toprovide a biological process for the large scale production of phenols.The inventors have developed a biological process that solves bothobjects.

The present invention thus provides in a first aspect a process for theproduction of a sulfated phenolic compound comprising:

(i′) contacting a medium comprising a phenolic compound with a firstrecombinant host cell; wherein the first recombinant host cell comprisesa heterologous polypeptide having an aryl sulfotransferase activity; or

(i″) contacting a medium comprising a fermentable carbon substrate witha first recombinant host cell; wherein the first recombinant host cellcomprises a heterologous polypeptide having an aryl sulfotransferaseactivity; or

(i′″) contacting a medium comprising a precursor of a phenolic compoundwith a first recombinant host cell; wherein the first recombinant hostcell comprises a heterologous polypeptide having an arylsulfotransferase activity.

The present invention provides in a further aspect a process for theproduction of a sulfated phenolic compound, such as zosteric acid, themethod comprises sulfating a phenolic compound, such as p-coumaric acid,using a polypeptide as detailed herein. Particularly, the processinvolves the use of a polypeptide having an aryl sulfotransferaseactivity, such as a polypeptide selected from the group consisting of:

a) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g. SEQ ID NO: 1);

b) a polypeptide comprising an amino acid sequence which has at leastabout 70%, such as at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 93%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1); or

c) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1),wherein 1 to 50, such as 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1to 15, 1 to 10, 1 to 5, 1 to 3, amino acid residues are substituted,deleted and/or inserted.

The present invention provides in a further aspect a recombinant hostcell as detailed herein. Particularly, the present invention provides arecombinant host cell comprising (e.g. expresses) a first heterologouspolypeptide having an aryl sulfotransferase activity, such as aheterologous polypeptide selected from the group consisting of:

a) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1);

b) a polypeptide comprising an amino acid sequence which has at leastabout 70%, such as at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 93%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1); or

c) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1),wherein 1 to 50, such as 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1to 15, 1 to 10, 1 to 5, 1 to 3, amino acid residues are substituted,deleted and/or inserted.

The present invention provides in a further aspect the use of apolypeptide as detailed herein in the sulfation of a phenolic compound.Particularly, the present invention provides the use of a polypeptidehaving an aryl sulfotransferase activity in the sulfation of a phenoliccompound, such as a polypeptide selected from the group consisting of:

a) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1);

b) a polypeptide comprising an amino acid sequence which has at leastabout 70%, such as at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 93%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1); or

c) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1),wherein 1 to 50, such as 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1to 15, 1 to 10, 1 to 5, 1 to 3, amino acid residues are substituted,deleted and/or inserted.

The present invention provides in a further aspect a compositioncomprising a first recombinant host cell as detailed herein and a secondrecombinant host cell as detailed herein. Particularly, the presentinvention provides a composition comprising a first recombinant hostcell comprising (e.g. expressing) a heterologous polypeptide having anaryl sulfotransferase activity, such as a heterologous polypeptideselected from the group consisting of:

a) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1);

b) a polypeptide comprising an amino acid sequence which has at leastabout 70%, such as at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 93%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1); or

c) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1),wherein 1 to 50, such as 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1to 15, 1 to 10, 1 to 5, 1 to 3, amino acid residues are substituted,deleted and/or inserted; and

a second recombinant host cell comprising (e.g., expressing) aheterologous polypeptide having tyrosine ammonia lyase activity, such asa polypeptide selected from the group consisting of:

d) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 (e.g., SEQ ID NO: 14);

e) a polypeptide comprising an amino acid sequence which has at leastabout 70%, such as at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 93%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 14, 15, 16,17, 18, 19, 20, 21, 22 or 23 (e.g., SEQ ID NO: 14); or

f) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 (e.g., SEQ ID NO: 14),wherein 1 to 50, such as 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1to 15, 1 to 10, 1 to 5, 1 to 3, amino acid residues are substituted,deleted and/or inserted.

The present invention provides in a further aspect a compositioncomprising a first polypeptide and a second polypeptide. Particularly,the present invention provides a composition comprising a firstpolypeptide having an aryl sulfotransferase activity, such as apolypeptide selected from the group consisting of:

a) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1);

b) a polypeptide comprising an amino acid sequence which has at leastabout 70%, such as at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 93%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1); or

c) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1),wherein 1 to 50, such as 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1to 15, 1 to 10, 1 to 5, 1 to 3, amino acid residues are substituted,deleted and/or inserted; and

a second polypeptide tyrosine ammonia lyase activity, such as apolypeptide selected from the group consisting of:

d) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 (e.g., SEQ ID NO: 14);

e) a polypeptide comprising an amino acid sequence which has at leastabout 70%, such as at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 93%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 14, 15, 16,17, 18, 19, 20, 21, 22 or 23 (e.g., SEQ ID NO: 14); or

f) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 (e.g., SEQ ID NO: 14),wherein 1 to 50, such as 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1to 15, 1 to 10, 1 to 5, 1 to 3, amino acid residues are substituted,deleted and/or inserted.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Map of plasmid for expression of SULT1A1 from Rattus norvegicusin Escherichia coli

FIG. 2: Map of plasmid for over-expression of cysDNC in E. coli.

FIG. 3: Map of plasmid for over-expression of cysDNCQ in E. coli.

FIG. 4: Map of plasmid for expression of RmXAL from Rhodotorulamucilaginosa/Rhodotorula rubra in E. coli.

FIG. 5: Toxicity of unsulfated or sulfated products

FIG. 6: Map of plasmid for expression of tyrosine ammonia-lyase RsTALfrom Rhodobacter sphaeroides in E. coli.

FIG. 7: Map of plasmid for expression of tyrosine ammonia-lyase FjTALfrom Flavobacterium johnsoniae in E. coli.

FIG. 8: Map of plasmid for expression of tyrosine ammonia-lyase RcTALfrom Rhodobacter capsulatus in E. coli.

FIG. 9: Map of plasmid for expression of SULT1A1 from Rattus norvegicusin Saccharomyces cerevisiae (native gene).

FIG. 10: Map of plasmid for expression of SULT1A1 from Rattus norvegicusin Saccharomyces cerevisiae (codon-optimized gene).

DETAILED DESCRIPTION OF THE INVENTION

Unless specifically defined herein, all technical and scientific termsused have the same meaning as commonly understood by a skilled artisanin the fields of biochemistry, genetics, and molecular biology.

All methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,with suitable methods and materials being described herein. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willprevail. Further, the materials, methods, and examples are illustrativeonly and are not intended to be limiting, unless otherwise specified.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, CurrentProtocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley andson Inc, Library of Congress, USA); Molecular Cloning: A LaboratoryManual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, NewYork: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis(M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; NucleicAcid Hybridization (B. D. Harries & S. J. Higgins eds. 1984);Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984);Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987);Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A PracticalGuide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J.Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York),specifically, Vols. 154 and 155 (Wu et al. eds.) and Vol. 185, “GeneExpression Technology” (D. Goeddel, ed.); Gene Transfer Vectors ForMammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold SpringHarbor Laboratory); Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987); Handbook OfExperimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1986).

Polypeptides and Host Cells

As indicated above, the present invention provides and utilizespolypeptides having aryl sulfotransferase activity (EC:2.8.2.1). Thismakes them particularly suitable for the sulfation of phenolic compoundssuch as p-coumaric acid and derivatives thereof (e.g., caffeic acid,ferulic acid or sinapic acid), or resveratrol.

The polypeptide having aryl sulfotransferase activity may be asulfotransferase 1A1 enzyme, a sulfotransferase 1A2 enzyme, asulfotransferase 1A3 enzyme, a sulfotransferase 1B1 enzyme, asulfotransferase 1C1 enzyme, a sulfotransferase 1C2 enzyme, asulfotransferase 1C4 enzyme, or a sulfotransferase 1E1 enzyme. Accordingto certain embodiments, the polypeptide having aryl sulfotransferaseactivity is a sulfotransferase 1A1 enzyme. According to certain otherembodiments, the polypeptide having aryl sulfotransferase activity is asulfotransferase 1A2 enzyme. According to certain embodiments, thepolypeptide having aryl sulfotransferase activity is a sulfotransferase1B1 enzyme. According to certain embodiments, the polypeptide havingaryl sulfotransferase activity is a sulfotransferase 1C1 enzyme.According to certain embodiments, the polypeptide having arylsulfotransferase activity is a sulfotransferase 1C2 enzyme. According tocertain embodiments, the polypeptide having aryl sulfotransferaseactivity is a sulfotransferase 1C4 enzyme. According to other certainembodiments, the polypeptide having aryl sulfotransferase activity is asulfotransferase 1E1 enzyme (estrogen sulfotransferase), such as thesulfotransferase 1E1 from Gallus gallus domesticus.

According to certain embodiments, the polypeptide having arylsulfotransferase activity is a mammalian aryl sulfotransferase, such asa mammalian sulfotransferase 1A1 enzyme.

According to certain embodiments, the polypeptide having arylsulfotransferase activity is an aryl sulfotransferase from Rattusnorvegicus or a variant thereof. Such variant may have at least about70%, such as at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 93%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, sequence identity to the aminoacid sequence of the aryl sulfotransferase from Rattus norvegicus. Suchvariant may also have an amino acid sequence of the sulfotransferasefrom Rattus norvegicus, wherein 1 to 50, such as 1 to 40, 1 to 35, 1 to30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, amino acidresidues are substituted, deleted and/or inserted.

It is understood that the foregoing values generally define the totalnumber of alterations to the reference aryl sulfotransferase. Thealterations may solely be amino acid substitutions, be it conserved ornon-conserved substitutions, or both. They may solely be amino aciddeletions. They may solely be amino acid insertions. The alterations maybe a mix of these specific alterations, such as amino acid substitutionsand amino acid insertions.

According to certain embodiments, the polypeptide having arylsulfotransferase activity may be a polypeptide selected from the groupconsisting of:

a) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1);

b) a polypeptide comprising an amino acid sequence which has at leastabout 70%, such as at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 93%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1); or

c) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1),wherein 1 to 50, such as 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1to 15, 1 to 10, 1 to 5, 1 to 3, amino acid residues are substituted,deleted and/or inserted.

According to certain embodiments, a polypeptide according to theinvention is a polypeptide according to a). Accordingly, a polypeptideaccording to the invention may be a polypeptide comprising an amino acidsequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12or 13 (e.g., SEQ ID NO: 1). According to particular embodiments, apolypeptide according to a) comprises an amino acid sequence set forthin SEQ ID NO: 1. According other particular embodiments, a polypeptideaccording to a) comprises an amino acid sequence set forth in SEQ ID NO:2. According to yet other particular embodiments, a polypeptideaccording to a) comprises an amino acid sequence set forth in SEQ ID NO:3. According to yet other particular embodiments, a polypeptideaccording to a) comprises an amino acid sequence set forth in SEQ ID NO:4. According to yet other particular embodiments, a polypeptideaccording to a) comprises an amino acid sequence set forth in SEQ ID NO:5. According other particular embodiments, a polypeptide according to a)comprises an amino acid sequence set forth in SEQ ID NO: 6. Accordingother particular embodiments, a polypeptide according to a) comprises anamino acid sequence set forth in SEQ ID NO: 7. According otherparticular embodiments, a polypeptide according to a) comprises an aminoacid sequence set forth in SEQ ID NO: 8. According other particularembodiments, a polypeptide according to a) comprises an amino acidsequence set forth in SEQ ID NO: 9. According other particularembodiments, a polypeptide according to a) comprises an amino acidsequence set forth in SEQ ID NO: 10. According other particularembodiments, a polypeptide according to a) comprises an amino acidsequence set forth in SEQ ID NO: 11. According other particularembodiments, a polypeptide according to a) comprises an amino acidsequence set forth in SEQ ID NO: 12. According other particularembodiments, a polypeptide according to a) comprises an amino acidsequence set forth in SEQ ID NO: 13.

According to other certain embodiments, a polypeptide according to theinvention is a polypeptide according to b). Accordingly, a polypeptideaccording to the invention may be a polypeptide comprising an amino acidsequence which has at least about 70%, such as at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, sequence identity to the amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1).According to particular embodiments, a polypeptide according to b)comprises an amino acid sequence which has at least about 80%, such asat least about 85%, at least about 90%, at least about 93%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity to the amino acid sequence setforth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g.,SEQ ID NO: 1). According to other particular embodiments, a polypeptideaccording to b) comprises an amino acid sequence which has at leastabout 85%, such as at least about 90%, at least about 93%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity to the amino acid sequence setforth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g.,SEQ ID NO: 1). According to other particular embodiments, a polypeptideaccording to b) comprises an amino acid sequence which has at leastabout 90%, such as at least about 93%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about99%, sequence identity to the amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1).According to other particular embodiments, a polypeptide according to b)comprises an amino acid sequence which has at least about 95%, such asat least about 96%, at least about 97%, at least about 98%, or at leastabout 99%, sequence identity to the amino acid sequence set forth in SEQID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1).

According to particular embodiments, a polypeptide according to b)comprises an amino acid sequence which has at least about 70%, such asat least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 93%, at least 95%, at least 96%, at least 97%,at least 98%, or at least 99%, sequence identity to the amino acidsequence set forth in SEQ ID NO: 1. According to more particularembodiments, a polypeptide according to b) comprises an amino acidsequence which has at least about 80%, such as at least about 85%, atleast about 90%, at least about 93%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%,sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.According to other more particular embodiments, a polypeptide accordingto b) comprises an amino acid sequence which has at least about 85%,such as at least about 90%, at least about 93%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99%, sequence identity to the amino acid sequence set forth in SEQID NO: 1. According to other more particular embodiments, a polypeptideaccording to b) comprises an amino acid sequence which has at leastabout 90%, such as at least about 93%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about99%, sequence identity to the amino acid sequence set forth in SEQ IDNO: 1. According to other more particular embodiments, a polypeptideaccording to b) comprises an amino acid sequence which has at leastabout 95%, such as at least about 96%, at least about 97%, at leastabout 98%, or at least about 99%, sequence identity to the amino acidsequence set forth in SEQ ID NO: 1.

Preferably, a polypeptide according to b) has aryl sulfotransferaseactivity. More preferably, a polypeptide according to b) has a arylsulfotransferase activity similar to that of the polypeptide comprisingan amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1).

According to certain embodiment, a polypeptide according to b) has arylsulfotransferase activity similar to that of the polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 1. According to certainother embodiments, a polypeptide according to b) has arylsulfotransferase activity similar to that of the polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 2. According to certainother embodiments, a polypeptide according to b) has arylsulfotransferase activity similar to that of the polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 3. According to certainother embodiments, a polypeptide according to b) has arylsulfotransferase activity similar to that of the polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 4. According to certainother embodiments, a polypeptide according to b) has arylsulfotransferase activity similar to that of the polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 5. According to certainother embodiments, a polypeptide according to b) has arylsulfotransferase activity similar to that of the polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 6. According to certainother embodiments, a polypeptide according to b) has arylsulfotransferase activity similar to that of the polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 7. According to certainother embodiments, a polypeptide according to b) has arylsulfotransferase activity similar to that of the polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 8. According to certainother embodiments, a polypeptide according to b) has arylsulfotransferase activity similar to that of the polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 9. According to certainother embodiments, a polypeptide according to b) has arylsulfotransferase activity similar to that of the polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 10. According to certainother embodiments, a polypeptide according to b) has arylsulfotransferase activity similar to that of the polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 11. According to certainother embodiments, a polypeptide according to b) has arylsulfotransferase activity similar to that of the polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 12. According to certainother embodiments, a polypeptide according to b) has arylsulfotransferase activity similar to that of the polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 13.

With “similar” aryl sulfotransferase activity, it is meant that thepolypeptide according to b) has at least about 10%, such as at leastabout 20%, at least about 30%, at least about 40%, at least about 50%,at least about 60, at least about 75%, at least about 80%, at leastabout 90%, at least about 95%, at least about 100%, at least about 200%,at least about 300%, at least about 400%, at least about 500%, at leastabout 800%, at least about 1000% or at least about 2000%, of the arylsulfotransferase activity of the reference polypeptide (e.g., SEQ ID NO:1).

The aryl sulfotransferase activity may for instance be determined inaccordance to the following method: Aryl sulfotransferase activity maybe determined by the reaction of radioactively sulfur labelled PAPS,[³⁵S]PAPS, with the substrate in presence of the polypeptide ofinterest. This is described previously, for example by Hattori et al(Biosci Biotechnol Biochem. 2008; 72(2):540-7). The reaction takes placein a buffer such as 250 μL 50 mM sodium phosphate pH 6.8 with 1 μM[³⁵S]PAPS (3.7 kBq) with 100 μM accepting compound for a period of 30min at 30° C. The reaction is stopped by addition of 100 μL of a 1:1mixture of 0.1 M barium acetate and barium hydroxide. 50 μL of 0.1 Mzinc sulfate is added, followed by centrifugation at 1,200×g for 5 min.300 μL of the supernatant is then transferred to a new container and 50μL of an equal volume of 0.1 M barium hydroxide and 0.1 M zinc sulfateis added. The mixture is then centrifuged at 13,000×g for 5 min, and300-μL aliquots of the supernatant are mixed with 2.5 mL of Cleasol I(Nacalai Tesque, Kyoto, Japan). The radioactivity is then measured byscintillation.

Alternatively, the activity of a sulfotransferase may be detected bydirect measurement of the product by analytical methods such as highperformance liquid chromatography (HPLC) or liquid chromatography incombination with mass spectrometry (LC-MS).

According to other certain embodiments, a polypeptide according to theinvention is a polypeptide according to c). Accordingly, a polypeptideaccording to the invention may be a polypeptide comprising an amino acidsequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12or 13 (e.g., SEQ ID NO: 1), wherein 1 or more, such as 2 or more, 3 ormore, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more,10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more,16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 25 or more,30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 60 or more,70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 ormore, 130 or more, 140 or more, or 150 or more, amino acid residues aresubstituted, deleted, and/or inserted. According to particularembodiments, a polypeptide according to c) comprises an amino acidsequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12or 13 (e.g., SEQ ID NO: 1), wherein about 1 to about 150, such as about1 to about 140, about 1 to about 130, about 1 to about 120, about 1 toabout 110, about 1 to about 100, about 1 to about 90, about 1 to about80, about 1 to about 70, about 1 to about 60, about 1 to about 50, about1 to about 40, about 1 to about 35, about 1 to about 30, about 1 toabout 25, about 1 to about 20, about 1 to about 15, about 1 to about 10,about 1 to about 5, or about 1 to about 3, amino acid residues aresubstituted, deleted and/or inserted. According to more particularembodiments, a polypeptide according to c) comprises an amino acidsequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12or 13 (e.g., SEQ ID NO: 1), wherein about 1 to about 50, about 1 toabout 40, about 1 to about 35, about 1 to about 30, about 1 to about 25,about 1 to about 20, about 1 to about 15, about 1 to about 10, about 1to about 5, or about 1 to about 3, amino acid residues are substituted,deleted and/or inserted. According to other more particular embodiments,a polypeptide according to c) comprises an amino acid sequence set forthin SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ IDNO: 1), wherein about 1 to about 30, such as about 1 to about 25, about1 to about 20, about 1 to about 15, about 1 to about 10, about 1 toabout 5, or about 1 to about 3, amino acid residues are substituted,deleted and/or inserted.

According to other more particular embodiments, a polypeptide accordingto c) comprises an amino acid sequence set forth in SEQ ID NO: 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1), wherein about 1to about 25, such as about 1 to about 20, about 1 to about 15, about 1to about 10, about 1 to about 5, or about 1 to about 3, amino acidresidues are substituted, deleted and/or inserted.

According to particular embodiments, a polypeptide according to c)comprises an amino acid sequence set forth in SEQ ID NO: 1, whereinabout 1 to about 150, such as about 1 to about 140, about 1 to about130, about 1 to about 120, about 1 to about 110, about 1 to about 100,about 1 to about 90, about 1 to about 80, about 1 to about 70, about 1to about 60, about 1 to about 50, about 1 to about 40, about 1 to about35, about 1 to about 30, about 1 to about 25, about 1 to about 20, about1 to about 15, about 1 to about 10, about 1 to about 5, or about 1 toabout 3, amino acid residues are substituted, deleted and/or inserted.According to more particular embodiments, a polypeptide according to c)comprises an amino acid sequence set forth in SEQ ID NO: 1, whereinabout 1 to about 50, about 1 to about 40, about 1 to about 35, about 1to about 30, about 1 to about 25, about 1 to about 20, about 1 to about15, about 1 to about 10, about 1 to about 5, or about 1 to about 3,amino acid residues are substituted, deleted and/or inserted. Accordingto other more particular embodiments, a polypeptide according to c)comprises an amino acid sequence set forth in SEQ ID NO: 1, whereinabout 1 to about 30, such as about 1 to about 25, about 1 to about 20,about 1 to about 15, about 1 to about 10, about 1 to about 5, or about 1to about 3, amino acid residues are substituted, deleted and/orinserted. According to other more particular embodiments, a polypeptideaccording to c) comprises an amino acid sequence set forth in SEQ ID NO:1, wherein about 1 to about 25, such as about 1 to about 20, about 1 toabout 15, about 1 to about 10, about 1 to about 5, or about 1 to about3, amino acid residues are substituted, deleted and/or inserted.

It is understood that the foregoing values generally define the totalnumber of alterations to the reference polypeptide (e.g., SEQ ID NO: 1).The alterations may solely be amino acid substitutions, be it conservedor non-conserved substitutions, or both. They may solely be amino aciddeletions. They may solely be amino acid insertions. The alterations maybe a mix of these specific alterations, such as amino acid substitutionsand amino acid insertions.

Preferably, a polypeptide according to c) has aryl sulfotransferaseactivity. More preferably, a polypeptide according to c) has a arylsulfotransferase activity similar to that of the polypeptide comprisingan amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1).

According to certain embodiment, a polypeptide according to c) has arylsulfotransferase activity similar to that of the polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 1. According to certainother embodiments, a polypeptide according to c) has arylsulfotransferase activity similar to that of the polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 2. According to certainother embodiments, a polypeptide according to c) has arylsulfotransferase activity similar to that of the polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 3. According to certainother embodiments, a polypeptide according to c) has arylsulfotransferase activity similar to that of the polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 4. According to certainother embodiments, a polypeptide according to c) has arylsulfotransferase activity similar to that of the polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 5. According to certainother embodiments, a polypeptide according to c) has arylsulfotransferase activity similar to that of the polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 6. According to certainother embodiments, a polypeptide according to c) has arylsulfotransferase activity similar to that of the polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 7. According to certainother embodiments, a polypeptide according to c) has arylsulfotransferase activity similar to that of the polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 8. According to certainother embodiments, a polypeptide according to c) has arylsulfotransferase activity similar to that of the polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 9. According to certainother embodiments, a polypeptide according to c) has arylsulfotransferase activity similar to that of the polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 10. According to certainother embodiments, a polypeptide according to c) has arylsulfotransferase activity similar to that of the polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 11. According to certainother embodiments, a polypeptide according to c) has arylsulfotransferase activity similar to that of the polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 12. According to certainother embodiments, a polypeptide according to c) has arylsulfotransferase activity similar to that of the polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 13.

With “similar” aryl sulfotransferase activity it is meant that thepolypeptide according to c) has at least about 10%, such as at leastabout 20%, at least about 30%, at least about 40%, at least about 50%,at least about 60, at least about 75%, at least about 80%, at leastabout 90%, at least about 95%, at least about 100%, at least about 200%,at least about 300%, at least about 400%, at least about 500%, at leastabout 800%, at least about 1000% or at least about 2000%, at of the arylsulfotransferase activity of the reference polypeptide (e.g., SEQ ID NO:1).

The aryl sulfotransferase activity may for instance be determined inaccordance to the following method: Aryl sulfotransferase activity maybe determined by the reaction of radioactively sulfur labelled PAPS,[³⁵S]PAPS, with the substrate in presence of the polypeptide ofinterest. This is described previously, for example by Hattori et al(Biosci Biotechnol Biochem. 2008; 72(2):540-7). The reaction takes placein a buffer such as 250 μL 50 mM sodium phosphate pH 6.8 with 1 μM[³⁵S]PAPS (3.7 kBq) with 100 μM accepting compound for a period of 30min at 30° C. The reaction is stopped by addition of 100 μL of a 1:1mixture of 0.1 M barium acetate and barium hydroxide. 50 μL of 0.1 Mzinc sulfate is added, followed by centrifugation at 1,200×g for 5 min.300 μL of the supernatant is then transferred to a new container and 50μL of an equal volume of 0.1 M barium hydroxide and 0.1 M zinc sulfateis added. The mixture is then centrifuged at 13,000×g for 5 min, and300-4 aliquots of the supernatant are mixed with 2.5 mL of Cleasol I(Nacalai Tesque, Kyoto, Japan). The radioactivity is then measured byscintillation.

Alternatively, the activity of a sulfotransferase may be detected bydirect measurement of the product by analytical methods such as highperformance liquid chromatography (HPLC) or liquid chromatography incombination with mass spectrometry (LC-MS).

Contemplated by the present invention is the production of a sulfatedphenolic compound from a precursor thereof, and in particular from aprecursor of the general formula (p-I) as described in more detailbelow. In this case, it may be suitable to employ a further (e.g.,second) polypeptide which has tyrosine ammonia lyase activity. Suchpolypeptide may be a polypeptide selected from the group consisting of:

d) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 (e.g., SEQ ID NO: 14);

e) a polypeptide comprising an amino acid sequence which has at leastabout 70%, such as at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 93%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99%, sequence identity to the amino acid sequence set forth in SEQID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 (e.g., SEQ ID NO: 14);or

f) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 (e.g., SEQ ID NO: 14),wherein 1 or more, such as about 1 to about 50, about 1 to about 40,about 1 to about 35, about 1 to about 30, about 1 to about 25, about 1to about 20, about 1 to about 15, about 1 to about 10, about 1 to about5, or about 1 to about 3, amino acid residues are substituted, deleted,and/or inserted.

According to certain embodiments, a further polypeptide according to theinvention is a polypeptide according to d). Accordingly, a polypeptideaccording to the invention may be a polypeptide comprising an amino acidsequence set forth in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or23 (e.g., SEQ ID NO: 14). According to particular embodiments, apolypeptide according to d) comprises an amino acid sequence set forthin SEQ ID NO: 14. According other particular embodiments, a polypeptideaccording to d) comprises an amino acid sequence set forth in SEQ ID NO:15. According to yet other particular embodiments, a polypeptideaccording to d) comprises an amino acid sequence set forth in SEQ ID NO:16. According other particular embodiments, a polypeptide according tod) comprises an amino acid sequence set forth in SEQ ID NO: 17.According other particular embodiments, a polypeptide according to d)comprises an amino acid sequence set forth in SEQ ID NO: 18. Accordingother particular embodiments, a polypeptide according to d) comprises anamino acid sequence set forth in SEQ ID NO: 19. According otherparticular embodiments, a polypeptide according to d) comprises an aminoacid sequence set forth in SEQ ID NO: 20. According other particularembodiments, a polypeptide according to d) comprises an amino acidsequence set forth in SEQ ID NO: 21. According other particularembodiments, a polypeptide according to d) comprises an amino acidsequence set forth in SEQ ID NO: 22. According other particularembodiments, a polypeptide according to d) comprises an amino acidsequence set forth in SEQ ID NO: 23.

According to other certain embodiments, a further polypeptide accordingto the invention is a polypeptide according to e). Accordingly, apolypeptide according to the invention may be a polypeptide comprisingan amino acid sequence which has at least about 70%, such as at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 93%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99%, sequence identity to the amino acid sequence setforth in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 (e.g., SEQID NO: 14). According to particular embodiments, a polypeptide accordingto e) comprises an amino acid sequence which has at least about 80%,such as at least about 85%, at least about 90%, at least about 93%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99%, sequence identity to the amino acid sequenceset forth in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 (e.g.,SEQ ID NO: 14). According to other particular embodiments, a polypeptideaccording to e) comprises an amino acid sequence which has at leastabout 85%, such as at least about 90%, at least about 93%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity to the amino acid sequence setforth in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 (e.g., SEQID NO: 14). According to other particular embodiments, a polypeptideaccording to e) comprises an amino acid sequence which has at leastabout 90%, such as at least about 93%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about99%, sequence identity to the amino acid sequence set forth in SEQ IDNO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 (e.g., SEQ ID NO: 14).According to other particular embodiments, a polypeptide according to e)comprises an amino acid sequence which has at least about 95%, such asat least about 96%, at least about 97%, at least about 98%, or at leastabout 99%, sequence identity to the amino acid sequence set forth in SEQID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 (e.g., SEQ ID NO: 14).

According to particular embodiments, a polypeptide according to e)comprises an amino acid sequence which has at least about 70%, such asat least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 93%, at least 95%, at least 96%, at least 97%,at least 98%, or at least 99%, sequence identity to the amino acidsequence set forth in SEQ ID NO: 14. According to more particularembodiments, a polypeptide according to e) comprises an amino acidsequence which has at least about 80%, such as at least about 85%, atleast about 90%, at least about 93%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%,sequence identity to the amino acid sequence set forth in SEQ ID NO: 14.According to other more particular embodiments, a polypeptide accordingto e) comprises an amino acid sequence which has at least about 85%,such as at least about 90%, at least about 93%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99%, sequence identity to the amino acid sequence set forth in SEQID NO: 14. According to other more particular embodiments, a polypeptideaccording to e) comprises an amino acid sequence which has at leastabout 90%, such as at least about 93%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about99%, sequence identity to the amino acid sequence set forth in SEQ IDNO: 14. According to other more particular embodiments, a polypeptideaccording to e) comprises an amino acid sequence which has at leastabout 95%, such as at least about 96%, at least about 97%, at leastabout 98%, or at least about 99%, sequence identity to the amino acidsequence set forth in SEQ ID NO: 14.

Preferably, a polypeptide according to e) has tyrosine ammonia lyaseactivity. More preferably, a polypeptide according to e) has a tyrosineammonia lyase activity similar to that of the polypeptide comprising anamino acid sequence set forth in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20,21, 22 or 23 (e.g., SEQ ID NO: 14).

According to certain embodiment, a polypeptide according to e) hastyrosine ammonia lyase activity similar to that of the polypeptidecomprising the amino acid sequence set forth in SEQ ID NO: 14. Accordingto certain other embodiments, a polypeptide according to e) has tyrosineammonia lyase activity similar to that of the polypeptide comprising theamino acid sequence set forth in SEQ ID NO: 15. According to certainother embodiments, a polypeptide according to e) has tyrosine ammonialyase activity similar to that of the polypeptide comprising the aminoacid sequence set forth in SEQ ID NO: 16. According to certain otherembodiments, a polypeptide according to e) has tyrosine ammonia lyaseactivity similar to that of the polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 17. According to certain otherembodiments, a polypeptide according to e) has tyrosine ammonia lyaseactivity similar to that of the polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 18. According to certain otherembodiments, a polypeptide according to e) has tyrosine ammonia lyaseactivity similar to that of the polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 19. According to certain otherembodiments, a polypeptide according to e) has tyrosine ammonia lyaseactivity similar to that of the polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 20. According to certain otherembodiments, a polypeptide according to e) has tyrosine ammonia lyaseactivity similar to that of the polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 21. According to certain otherembodiments, a polypeptide according to e) has tyrosine ammonia lyaseactivity similar to that of the polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 22. According to certain otherembodiments, a polypeptide according to e) has tyrosine ammonia lyaseactivity similar to that of the polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 23. With “similar” tyrosine ammonialyase activity it is meant that the polypeptide according to e) has atleast about 10%, such as at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60, at least about75%, at least about 80%, at least about 90%, at least about 95%, atleast about 100%, at least about 200%, at least about 300%, at leastabout 400%, at least about 500%, at least about 800%, at least about1000% or at least about 2000%, of the ammonia lyase activity of thereference polypeptide (e.g., SEQ ID NO: 14).

The tyrosine ammonia lyase activity may for instance be determined inaccordance to the following method: Enzymatic assays are performed in200 μL volumes in wells in a UV transparent 96-well plate, by followingthe increase in absorbance at 315 nm (pHCA) using spectrophotometry orHPLC with UV detection. The reaction mixtures contain 2 μg of purifiedprotein and are initiated by adding 1 mM tyrosine or 6 mM afterequilibration to 30° C. The enzymatic activity is calculated as U/g,where U is defined as μmol substrate converted per minute. Negativecontrols contain no purified protein. Kinetic constants Km and vmax aredetermined from assays containing 1.56 μM to 200 μM tyrosine.

According to other certain embodiments, a further polypeptide accordingto the invention is a polypeptide according to f). Accordingly, apolypeptide according to the invention may be a polypeptide comprisingan amino acid sequence set forth in SEQ ID NO: 14, 15, 16, 17, 18, 19,20, 21, 22 or 23 (e.g., SEQ ID NO: 14), wherein 1 or more, such as 2 ormore, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more,9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more,15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more,25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more,60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 ormore, 120 or more, 130 or more, 140 or more, or 150 or more, amino acidresidues are substituted, deleted, and/or inserted. According toparticular embodiments, a polypeptide according to f) comprises an aminoacid sequence set forth in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22or 23 (e.g., SEQ ID NO: 14), wherein about 1 to about 150, such as about1 to about 140, about 1 to about 130, about 1 to about 120, about 1 toabout 110, about 1 to about 100, about 1 to about 90, about 1 to about80, about 1 to about 70, about 1 to about 60, about 1 to about 50, about1 to about 40, about 1 to about 35, about 1 to about 30, about 1 toabout 25, about 1 to about 20, about 1 to about 15, about 1 to about 10,about 1 to about 5, or about 1 to about 3, amino acid residues aresubstituted, deleted and/or inserted. According to more particularembodiments, a polypeptide according to f) comprises an amino acidsequence set forth in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or23 (e.g., SEQ ID NO: 14), wherein about 1 to about 50, about 1 to about40, about 1 to about 35, about 1 to about 30, about 1 to about 25, about1 to about 20, about 1 to about 15, about 1 to about 10, about 1 toabout 5, or about 1 to about 3, amino acid residues are substituted,deleted and/or inserted. According to other more particular embodiments,a polypeptide according to f) comprises an amino acid sequence set forthin SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 (e.g., SEQ ID NO:14), wherein about 1 to about 30, such as about 1 to about 25, about 1to about 20, about 1 to about 15, about 1 to about 10, about 1 to about5, or about 1 to about 3, amino acid residues are substituted, deletedand/or inserted. According to other more particular embodiments, apolypeptide according to f) comprises an amino acid sequence set forthin SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 (e.g., SEQ ID NO:14), wherein about 1 to about 25, such as about 1 to about 20, about 1to about 15, about 1 to about 10, about 1 to about 5, or about 1 toabout 3, amino acid residues are substituted, deleted and/or inserted.

According to particular embodiments, a polypeptide according to f)comprises an amino acid sequence set forth in SEQ ID NO: 14, whereinabout 1 to about 150, such as about 1 to about 140, about 1 to about130, about 1 to about 120, about 1 to about 110, about 1 to about 100,about 1 to about 90, about 1 to about 80, about 1 to about 70, about 1to about 60, about 1 to about 50, about 1 to about 40, about 1 to about35, about 1 to about 30, about 1 to about 25, about 1 to about 20, about1 to about 15, about 1 to about 10, about 1 to about 5, or about 1 toabout 3, amino acid residues are substituted, deleted and/or inserted.According to more particular embodiments, a polypeptide according to f)comprises an amino acid sequence set forth in SEQ ID NO: 14, whereinabout 1 to about 50, about 1 to about 40, about 1 to about 35, about 1to about 30, about 1 to about 25, about 1 to about 20, about 1 to about15, about 1 to about 10, about 1 to about 5, or about 1 to about 3,amino acid residues are substituted, deleted and/or inserted. Accordingto other more particular embodiments, a polypeptide according to f)comprises an amino acid sequence set forth in SEQ ID NO: 14, whereinabout 1 to about 30, such as about 1 to about 25, about 1 to about 20,about 1 to about 15, about 1 to about 10, about 1 to about 5, or about 1to about 3, amino acid residues are substituted, deleted and/orinserted. According to other more particular embodiments, a polypeptideaccording to f) comprises an amino acid sequence set forth in SEQ ID NO:14, wherein about 1 to about 25, such as about 1 to about 20, about 1 toabout 15, about 1 to about 10, about 1 to about 5, or about 1 to about3, amino acid residues are substituted, deleted and/or inserted.

It is understood that the foregoing values generally define the totalnumber of alterations to the reference polypeptide (e.g., SEQ ID NO:14). The alterations may solely be amino acid substitutions, be itconserved or non-conserved substitutions, or both. They may solely beamino acid deletions. They may solely be amino acid insertions. Thealterations may be a mix of these specific alterations, such as aminoacid substitutions and amino acid insertions.

Preferably, a polypeptide according to f) has tyrosine ammonia lyaseactivity. More preferably, a polypeptide according to f) has a tyrosineammonia lyase activity similar to that of the polypeptide comprising anamino acid sequence set forth in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20,21, 22 or 23 (e.g., SEQ ID NO: 14). According to certain embodiment, apolypeptide according to f) has tyrosine ammonia lyase activity similarto that of the polypeptide comprising the amino acid sequence set forthin SEQ ID NO: 14. According to certain other embodiments, a polypeptideaccording to f) has tyrosine ammonia lyase activity similar to that ofthe polypeptide comprising the amino acid sequence set forth in SEQ IDNO: 15. According to certain other embodiments, a polypeptide accordingto f) has tyrosine ammonia lyase activity similar to that of thepolypeptide comprising the amino acid sequence set forth in SEQ ID NO:16. According to certain other embodiments, a polypeptide according tof) has tyrosine ammonia lyase activity similar to that of thepolypeptide comprising the amino acid sequence set forth in SEQ ID NO:17. According to certain other embodiments, a polypeptide according tof) has tyrosine ammonia lyase activity similar to that of thepolypeptide comprising the amino acid sequence set forth in SEQ ID NO:18. According to certain other embodiments, a polypeptide according tof) has tyrosine ammonia lyase activity similar to that of thepolypeptide comprising the amino acid sequence set forth in SEQ ID NO:19. According to certain other embodiments, a polypeptide according tof) has tyrosine ammonia lyase activity similar to that of thepolypeptide comprising the amino acid sequence set forth in SEQ ID NO:20. According to certain other embodiments, a polypeptide according tof) has tyrosine ammonia lyase activity similar to that of thepolypeptide comprising the amino acid sequence set forth in SEQ ID NO:21. According to certain other embodiments, a polypeptide according tof) has tyrosine ammonia lyase activity similar to that of thepolypeptide comprising the amino acid sequence set forth in SEQ ID NO:22. According to certain other embodiments, a polypeptide according tof) has tyrosine ammonia lyase activity similar to that of thepolypeptide comprising the amino acid sequence set forth in SEQ ID NO:23. With “similar” tyrosine ammonia lyase activity it is meant that thepolypeptide according to f) has at least about 10%, such as at leastabout 20%, at least about 30%, at least about 40%, at least about 50%,at least about 60, at least about 75%, at least about 80%, at leastabout 90%, at least about 95%, at least about 100%, at least about 200%,at least about 300%, at least about 400%, at least about 500%, at leastabout 800%, at least about 1000% or at least about 2000%, of the ammonialyase activity of the reference polypeptide (e.g., SEQ ID NO: 14).

The tyrosine ammonia lyase activity may for instance be determined inaccordance to the following method: Enzymatic assays are performed in200 μL volumes in wells in a UV transparent 96-well plate, by followingthe increase in absorbance at 315 nm (pHCA) using spectrophotometry orHPLC with UV detection. The reaction mixtures contain 2 μg of purifiedprotein and are initiated by adding 1 mM tyrosine or 6 mM afterequilibration to 30° C. The enzymatic activity is calculated as U/g,where U is defined as μmol substrate converted per minute. Negativecontrols contain no purified protein. Kinetic constants Km and vmax aredetermined from assays containing 1.56 μM to 200 μM tyrosine.

Further contemplated by the present invention is to employ a further(e.g., third) polypeptide which has phenylalanine ammonia lyaseactivity, such as a phenylalanine ammonia lyase (EC 4.3.1.24).

The polypeptides may be employed in accordance with the invention inisolated form, such as in purified form. The polypeptides may forinstance be expressed by a recombinant host cell, and then purified.Techniques and means for the purification of polypeptides produced by arecombinant host cell are well know in the art. For example, in order tofacilitate purification, an amino acid motif comprising severalhistidine residues, such as at least 6, may be inserted at the C- orN-terminal end of the polypeptide. A non-limiting example of such aminoacid motif is provided in SEQ ID NO: 24. Various purification kits forhistidine-tagged polypeptides are available from commercial sources suchas Qiagen, Hilden, Germany; Clontech, Mountain View, Calif., USA;Bio-Rad, Hercules, Calif., USA and others.

Alternatively, the polypeptides may be chemically synthezised.Techniques for chemical peptide synthesis are well know and includeLiquid-phase synthesis and Solid-phase synthesis.

The polypeptides can also be employed in accordance with the inventionas part of a recombinant host cell. Such recombinant host cells aredescribed in more details below.

It is understood that the details given herein with respect topolypeptides apply to all aspects of the invention.

The present invention also provides recombinant host cells comprising(e.g. expressing) one or more polypeptides as detailed herein.Generally, the polypeptides according to the invention will beheterologous to the host cells, which means that the polypeptides arenormally not found in or made (i.e. expressed) by the host cells, butderived from a different species.

Therefore, the present invention provides a recombinant host cellcomprising a heterologous polypeptide having an aryl sulfotransferaseactivity. According to certain embodiments, a recombinant host cellaccording to the invention comprises a heterologous polypeptide selectedfrom the group consisting of:

a) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1);

b) a polypeptide comprising an amino acid sequence which has at leastabout 70%, such as at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 93%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1); or

c) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1),wherein 1 to 50, such as 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1to 15, 1 to 10, 1 to 5, 1 to 3, amino acid residues are substituted,deleted and/or inserted.

A recombinant host cell provided and utilized in accordance with thepresent invention may comprise a hetereologus polypeptide havingtyrosine ammonia lyase activity. According to certain embodiments, arecombinant host cell according to the invention comprises aheterologous polypeptide selected from the group consisting of:

d) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 (e.g., SEQ ID NO: 14);

e) a polypeptide comprising an amino acid sequence which has at leastabout 70%, such as at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 93%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 14, 15, 16,17, 18, 19, 20, 21, 22 or 23 (e.g., SEQ ID NO: 14); or

f) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 (e.g., SEQ ID NO: 14),wherein 1 or more, such as about 1 to about 50, about 1 to about 40,about 1 to about 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to5, 1 to 3, amino acid residues are substituted, deleted and/or inserted.

According to certain embodiments, a recombinant host cell comprises afirst heterologous polypeptide having aryl sulfotransferase activity anda second heterologous polypeptide having tyrosine ammonia lyaseactivity. According to particular embodiments, a recombinant host cellcomprises a first heterologous polypeptide selected from thepolypeptides according to items a) to c) as detailed herein, and asecond heterologous polypeptide selected from the polypeptides accordingto items e) to f) as detailed herein.

According to more particular embodiments, a recombinant host cellcomprises a first heterologous polypeptide selected from the groupconsisting of:

a) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1;

b) a polypeptide comprising an amino acid sequence which has at leastabout 70%, such as at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 93%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 1; or

c) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, wherein 1 to 50, such as 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, amino acid residues aresubstituted, deleted and/or inserted; and a second heterologouspolypeptide selected from the group consisting of:

d) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 14;

e) a polypeptide comprising an amino acid sequence which has at leastabout 70%, such as at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 93%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 14; or

f) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 14, wherein 1 to 50, such as 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, amino acid residues aresubstituted, deleted and/or inserted.

Alternatively, the first heterologous polypeptide having arylsulfotransferase activity may be comprised by a first recombinant hostcell, and the second heterologous polypeptide having tyrosine ammonialyase activity may be comprised by a second recombinant host cell.

According to certain embodiments, a recombinant host cell comprises afirst heterologous polypeptide having aryl sulfotransferase activity anda further (e.g., third) heterologous polypeptide having phenylalanineammonia lyase activity.

Alternatively, the first heterologous polypeptide having arylsulfotransferase activity may be comprised by a first recombinant hostcell, and the further (e.g., third) heterologous polypeptide havingphenylalanine ammonia lyase activity may be comprised by a further(e.g., third) recombinant host cell. Such further recombinant host cellmay be a recombinant host cell also comprising a heterologouspolypeptide having tyrosine ammonia lyase activity.

Recombinant host cells in accordance with the invention can be producedfrom any suitable host organism, including single-celled ormulticellular microorganisms such as bacteria, yeast, fungi, algae andplant, and higher eukaryotic organisms including nematodes, insects,reptiles, birds, amphibians and mammals.

According to certain embodiments, a recombinant host cells in accordancewith the invention is selected from the group consisting of bacteria,yeast, fungi, algae and plant.

According to certain other embodiments, a recombinant host cells inaccordance with the invention is selected from the group consisting ofbacteria, yeast, fungi, and algae.

According to certain other embodiments, a recombinant host cells inaccordance with the invention is selected from the group consisting ofbacteria, yeast and fungi.

According to certain other embodiments, a recombinant host cells inaccordance with the invention is selected from the group consisting ofbacteria and yeast.

According to certain embodiments, a recombinant host cells in accordancewith the invention is not a plant cell.

Bacterial host cells are selected from Gram-positive and Gram-negativebacteria. Non-limiting examples for Gram-negative bacterial host cellsinclude species from the genera Escherichia, Erwinia, Klebsiella andCitrobacter. Non-limiting examples of Gram-positive bacterial host cellsinclude species from the genera Bacillus, Lactococcus, Lactobacillus,Clostridium, Corynebacterium, Streptomyces, Streptococcus, andCellulomonas.

According to certain embodiments, the recombinant host cell is abacterium, which may be a bacterium of the genus Bacillus, Lactococcus,Lactobacillus, Clostridium, Corynebacterium, Geobacillus,Thermoanaerobacterium, Streptococcus, Pseudomonas, Streptomyces,Escherichia, Shigella, Acinetobacter, Citrobacter, Salmonella,Klebsiella, Enterobacter, Erwinia, Kluyvera, Serratia, Cedecea,Morganella, Hafnia, Edwardsiella, Providencia, Proteus, or Yersinia.

According to particular embodiments, the recombinant host cell is abacterium of the genus Bacillus. Non-limiting examples of a bacteria ofthe genus Bacillus are Bacillus subtitlis, Bacillus amyloliquefaciens,Bacillus licheniformis, and Bacillus mojavensis. According to moreparticular embodiments, the recombinant host cell is Bacillus subtitlis.According to other more particular embodiments, the recombinant hostcell is Bacillus licheniformis.

According to other particular embodiments, the recombinant host cell isa bacterium of the genus Lactococcus. A non-limiting example of abacterium of the genus Lactococcus is Lactococcus lactis. According tomore particular embodiments, the recombinant host cell is Lactococcuslactis.

According to other particular embodiments, the recombinant host cell isa bacterium of the genus Corynebacterium. A non-limiting example of abacterium of the genus Corynebacterium is Corynebacterium glutamicum.According to more particular embodiments, the recombinant host cell isCorynebacterium glutamicum.

According to other particular embodiments, the recombinant host cell isa bacterium of the genus Streptomyces. A non-limiting examples of abacterium of the genus Streptomyces are Streptomyces lividans,Streptomyces coelicolor, or Streptomyces griseus. According to moreparticular embodiments, the recombinant host cell is Streptomyceslividans. According to other more particular embodiments, therecombinant host cell is Streptomyces coelicolor. According to othermore particular embodiments, the recombinant host cell is Streptomycesgriseus.

According to other particular embodiments, the recombinant host cell isa bacterium of the genus Pseudomonas. A non-limiting example of abacterium of the genus Pseudomonas is Pseudomonas putida. According tomore particular embodiments, the recombinant host cell is Pseudomonasputida.

According to other particular embodiments, the recombinant host cell isa bacterium of the genus Geobacillus. A non-limiting examples of abacterium of the genus Geobacillus are Geobacillus thermoglucosidasiusand Geobacillus stearothermophilus. According to more particularembodiments, the recombinant host cell is Geobacillusthermoglucosidasius. According to other more particular embodiments, therecombinant host cell is Geobacillus stearothermophilus.

According to other particular embodiments, the recombinant host cell isa bacterium of the genus Thermoanaerobacterium. A non-limiting exampleof a bacterium of the genus Pseudomonas is Thermoanaerobacteriumthermosaccharolyticum. According to more particular embodiments, therecombinant host cell is Thermoanaerobacterium thermosaccharolyticum.

According to other particular embodiments, the recombinant host cell isa bacterium of the genus Escherichia. A non-limiting example of abacterium of the genus Escherichia is Escherichia coli. According tomore particular embodiments, the recombinant host cell is Escherichiacoli.

Yeast host cells may be derived from e.g., Saccharomyces, Pichia,Schizosacharomyces, Zygosaccharomyces, Hansenula, Pachyosolen,Kluyveromyces, Debaryomyces, Yarrowia, Candida, Cryptococcus,Komagataella, Lipomyces, Rhodospiridium, Rhodotorula, or Trichosporon.

According to certain embodiments, the recombinant host cell is a yeast,which may be a yeast is of the genus Saccharomyces, Pichia,Schizosacharomyces, Zygosaccharomyces, Hansenula, Pachyosolen,Kluyveromyces, Debaryomyces, Yarrowia, Candida, Cryptococcus,Komagataella, Lipomyces, Rhodospiridium, Rhodotorula, or Trichosporon.

According to particular embodiments, the recombinant host cell is ayeast of the genus Saccharomyces. A non-limiting example of a yeast ofthe genus Saccharomyces is Saccharomyces cerevisiae. According to moreparticular embodiments, the recombinant host cell is Saccharomycescerevisiae.

According to particular embodiments, the recombinant host cell is ayeast of the genus Pichia. Non-limiting example of a yeast of the genusPichia are Pichia pastoris and pichia kudriavzevii. According to moreparticular embodiments, the recombinant host cell is Pichia pastoris.According to other more particular embodiments, the recombinant hostcell is pichia kudriavzevii.

Fungi host cells may be derived from, e.g., Aspergillus.

According to certain embodiments, the recombinant host cell is a fungus,such as a fungi of the genus Aspergillus. Non-limiting examples of afungus of the genus Aspergillus are Aspergillus Oryzae, Aspergillusniger or Aspergillus awamsii. According to more particular embodiments,the recombinant host cell is Aspergillus Oryzae. According to other moreparticular embodiments, the recombinant host cell is Aspergillus niger.According to other more particular embodiments, the recombinant hostcell is Aspergillus awamsii.

Algae host cells may be derived from, e.g., Chlamydomonas,Haematococcus, Phaedactylum, Volvox or Dunaliella.

According to certain embodiments, the recombinant host cell is an alga,which may be an algae of the genus Chlamydomonas, Haematococcus,Phaedactylum, Volvox or Dunaliella.

According to particular embodiments, the recombinant host cell is analga cell of the genus Chlamydomonas. A non-limiting example of an algaof the genus Chlamydomonas is Chlamydomonas reinhardtii.

According to particular embodiments, the recombinant host cell is analga cell of the genus Haematococcus. A non-limiting example of an algaof the genus Haematococcus is Haematococcus pluvialis.

According to other particular embodiments, the recombinant host cell isan alga cell of the genus Phaedactylum. A non-limiting example of analga of the genus Phaedactylum is Phaedactylum tricornatum.

A plant host cell may be derived from, e.g., soybean, rapeseed,sunflower, cotton, corn, tobacco, alfalfa, wheat, barley, oats, sorghum,lettuce, rice, broccoli, cauliflower, cabbage, parsnips, melons,carrots, celery, parsley, tomatoes, potatoes, strawberries, peanuts,grapes, grass seed crops, sugar beets, sugar cane, beans, peas, rye,flax, hardwood trees, softwood trees, and forage grasses.

According to certain embodiments, the recombinant host cell is a plantcell, such as a plant cell selected from the group consisting ofsoybean, rapeseed, sunflower, cotton, corn, tobacco, alfalfa, wheat,barley, oats, sorghum, lettuce, rice, broccoli, cauliflower, cabbage,parsnips, melons, carrots, celery, parsley, tomatoes, potatoes,strawberries, peanuts, grapes, grass seed crops, sugar beets, sugarcane, beans, peas, rye, flax, hardwood trees, softwood trees, and foragegrasses.

Generally, a recombinant host cell according to the invention has beengenetically modified to express one or more polypeptides as detailedherein, which means that one or more exogenous nucleic acid molecules,such as DNA molecules, which comprise(s) a nucleotide sequence ornucleotide sequences encoding said polypeptide or polypeptides has beenintroduced in the host cell. Techniques for introducing exogenousnucleic acid molecule, such as a DNA molecule, into the various hostcells are well-known to those of skill in the art, and includetransformation (e.g., heat shock or natural transformation),transfection, conjugation, electroporation, microinjection andmicroparticle bombardment.

Accordingly, a recombinant host cell according to the inventioncomprises an exogenous nucleic acid molecule comprising a nucleotidesequence encoding a polypeptide as detailed herein.

In order to facilitate expression of the polypeptide in the host cell,the exogenous nucleic acid molecule may comprise suitable regulatoryelements such as a promoter that is functional in the host cell to causethe production of an mRNA molecule and that is operably linked to thenucleotide sequence encoding said polypeptide.

Promoters useful in accordance with the invention are any knownpromoters that are functional in a given host cell to cause theproduction of an mRNA molecule. Many such promoters are known to theskilled person. Such promoters include promoters normally associatedwith other genes, and/or promoters isolated from any bacteria, yeast,fungi, alga or plant cell. The use of promoters for protein expressionis generally known to those of skilled in the art of molecular biology,for example, see Sambrook et al., Molecular cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989.The promoter employed may be inducible. The term “inducible” used in thecontext of a promoter means that the promoter only directs transcriptionof an operably linked nucleotide sequence if a stimulus is present, suchas a change in temperature or the presence of a chemical substance(“chemical inducer”). As used herein, “chemical induction” according tothe present invention refers to the physical application of a exogenousor endogenous substance (incl. macromolecules, e.g., proteins or nucleicacids) to a host cell. This has the effect of causing the targetpromoter present in the host cell to increase the rate of transcription.Alternatively, the promoter employed may be constitutive. The term“constitutive” used in the context of a promoter means that the promoteris capable of directing transcription of an operably linked nucleotidesequence in the absence of stimulus (such as heat shock, chemicalsetc.).

Non-limiting examples of promoters functional in bacteria, such asBacillus subtilis, Lactococcus lactis or Escherichia coli, include bothconstitutive and inducible promoters such as T7 promoter, thebeta-lactamase and lactose promoter systems; alkaline phosphatase (phoA)promoter, a tryptophan (trp) promoter system, tetracycline promoter,lambda-phage promoter, ribosomal protein promoters; and hybrid promoterssuch as the tac promoter. Other bacterial and synthetic promoters arealso suitable.

Non-limiting examples of promoters functional in yeast, such asSaccharomyces cerevisiae, include xylose promoter, GAL1 and GAL10promoters, TEF1 promoter, and pgk1 promoter.

Non-limiting examples of promoters functional in fungi, such asAspergillus Oryzae or Aspergillus niger, include promotors derived fromthe gene encoding Aspergillus oryzae TAKA amylase, Aspergillus nigerneutral α-amylase, Aspergillus niger acid stable α-amylase, Aspergillusniger or Aspergillus awamsii glucoamylase (gluA), Aspergillus nigeracetamidase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphatase isomerase, Rhizopus meihei aspartic proteinase, andRhizopus meihei lipase.

Non-limiting examples of promoters functional in alga, such asHaematococcus pluvialis, include the CaMV35S promoter, the SV40promoter, and promoter of the Chlamydomonas reinhardtii RBCS2 gene andthe promoter of the Volvox carteri ARS gene.

Non-limiting examples of promoters functional in plant cells include theLactuca sative psbA promoter, the tabacco psbA promoter, the tobaccorrn16 PEP+NEP promoter, the CaMV 35S promoter, the 19S promoter, thetomate E8 promoter, the nos promoter, the Mac promoter, and the pet Epromoter or the ACT1 promoter.

Besides a promoter, the exogenous nucleic acid molecule may furthercomprise at least one regulatory element selected from a 5′ untranslatedregion (5′UTR) and 3′ untranslated region (3′ UTR). Many such 5′ UTRsand 3′ UTRs derived from prokaryotes and eukaryotes are well known tothe skilled person. Such regulatory elements include 5′ UTRs and 3′ UTRsnormally associated with other genes, and/or 5′ UTRs and 3′ UTRsisolated from any bacteria, yeast, fungi, alga or plant cell.

If the host cell is a prokaryotic organism, the 5′ UTR usually containsa ribosome binding site (RBS), also known as the Shine Dalgarno sequencewhich is usually 3-10 base pairs upstream from the initiation codon.Meanwhile, if the host cell is an eukaryotic organism the 5′ UTR usuallycontains the Kozak consensus sequence. An eukaryotic 5′ UTR may alsocontain cis-acting regulatory elements.

The exogenous nucleic acid molecule may be a vector or part of a vector,such as an expression vector. Normally, such a vector remainsextrachromosomal within the host cell which means that it is foundoutside of the nucleus or nucleoid region of the host cell.

According to certain embodiments, a recombinant host cell according tothe invention does not express an endogenous PAPS-dependent arylsulfotransferase.

It is also contemplated by the present invention that the exogenousnucleic acid molecule is stably integrated into the genome of the hostcell. Means for stable integration into the genome of a host cell, e.g.,by homologous recombination, are well known to the skilled person.

The sulfation reaction depends on the supply of sulfate from3′-phosphoadenosine 5′-phosphosulfate (PAPS) or transferred from anothersulfated compound. The inventors have shown that the sulfation reactioncan be enhanced by improving the supply of PAPS (3′-phosphoadenosine5′-phosphosulfate) and, in addition, by the removal of the product3′-phosphoadenosine 5′-phosphate (PAP). The improved supply is obtainedby deregulation, mutation or overexpression of enzymes that increasePAPS concentration or similarly reduce PAP concentration. This isexemplified in Example 2, where an increased production of zosteric acidin Escherichia coli is obtained by increasing the expression of thegenes cysD, cysN, and cysC which are responsible for production of PAPS.Without being bound to a specific theory, it is believed that anadenylyl moiety (AMP) of ATP is transferred to sulfate to form activatedsulfate, or APS (adenosine 5′-phosphosulfate). This extremelyunfavorable reaction is kinetically and energetically linked to thehydrolysis of GTP by the enzyme ATP sulfurylase, which is composed oftwo types of subunits: an adenylyl transferase (cysD) and a GTPase(cysN). APS is then phosphorylated at the 3′-hydroxyl to form PAPS(3′-phosphoadenosine 5′-phosphosulfate) in a reaction catalysed by APSkinase, which is encoded by cysC. Furthermore, the inventors haveenhanced the production of zosteric acid even more by increasing theexpression of the gene cysQ encoding a PAP phosphatase which isresponsible for the removal of PAP.

Therefore, in order to further improve the production of a sulfatedphenolic compound, such as zosteric acid, a recombinant host cellaccording to the present invention may be further modified to have anincreased protein expression of an ATP sulfurylase compared to anidentical host cell that does not carry said modification; may befurther modified to have an increased protein expression of an APSkinase compared to an identical host cell that does not carry saidmodification; and/or may be further modified to have an increasedprotein expression of a PAP phosphatase compared to an identical hostcell that does not carry said modification. By “increased proteinexpression” it is meant that the amount of the respective proteinproduced by the thus modified host cell is increased compared anidentical host cell that does not carry said modification. Moreparticularly, by “increase expression” it is meant that the amount ofrespective protein produced by the thus modified host cell is increasedby at least 10%, such as at least 20%, at least 30%, at least 40%, atleast 50% at least 60%, at least 70%, at least 80%, at least 90%, atleast 100%, at least 150%, at least 200%, at least 300%, at least 400%,at least 500%, at least 600%, at least 700% at least 800%, at leastabout 900%, at least about 1000%, at least about 2000%, at least about3000%, at least about 4000%, at least about 5000%, at least about 6000%,at least about 7000%, at least about 8000% at least about 9000% or atleast about 10000%, compared an identical host cell that does not carrysaid modification. The amount of protein in a given cell can bedetermined by any suitable quantification technique known in the art,such as ELISA, Immunohistochemistry or Western Blotting.

According to certain embodiments, a recombinant host cell according tothe invention has further been modified to have an increased proteinexpression an ATP sulfurylase compared to an identical host cell thatdoes not carry said modification.

According to certain embodiments, a recombinant host cell according tothe invention has further been modified to have an increased proteinexpression of an APS kinase compared to an identical host cell that doesnot carry said modification.

According to certain embodiments, a recombinant host cell according tothe invention has further been modified to have an increased proteinexpression of a PAP phosphatase compared to an identical host cell thatdoes not carry said modification.

An increase in protein expression may be achieved by any suitable meanswell-know to those skilled in the art. For example, an increase inprotein expression may be achieved by increasing the number of copies ofthe gene or genes encoding the respective protein (e.g., ATPsulfurylase, APS kinase and/or PAP phosphatase) in the host cell, suchas by using (e.g., introducing into the host cell) a vectors comprisingthe gene or genes operably linked to a promoter that is functional inthe host cell to cause the production of an mRNA molecule. An increasein protein expression may also be achieved by integration of at least asecond copy of the gene or genes encoding the respective protein intothe genome of the host cell. An increase in protein expression may alsobe achieved by increasing the strength of the promoter(s) operablylinked to the gene or genes. An increase in protein expression may alsobe achieved by modifying the ribosome binding site on the mRNA moleculeencoding the respective protein (e.g., ATP sulfurylase, APS kinaseand/or PAP phosphatase). By modifying the sequence of the ribosomebinding site the translation initiation rate may be increased, thusincreasing the translation efficiency.

ATP sulfurylase encoding genes for use according to the invention mayfor instance be the cysD and cysN genes from Escherichia coli (encodingSEQ ID NO: 25 and 26, respectively). Alternative ATP sulfurylaseencoding genes include the Arabidopsis thaliana ATP sulfurylase ASALgene (GenBank Accession No. U40715, Logan et al. (1996) J Biol Chem 271:12227); the Allium cepa ATP-sulfurylase gene (Gen-Bank Accession NoAF21154); the Lotus japonicus ATP sulfurylase gene (GenBank AccessionNo. AW164083); the Arabidopsis thaliana met3-1 ATP sulfurylase gene(Gen-Bank Accession No. X79210).

According to certain embodiments, a recombinant host cell according tothe invention comprises an exogenous nucleic acid molecule (such as avector) comprising one or more nucleotide sequences encoding a ATPsulfurylase.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such as avector) comprising a nucleotide sequence encoding i) a polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 25 or ii) apolypeptide comprising an amino acid sequence which has at least about70%, such as at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 93%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, sequence identity to the aminoacid sequence set forth in SEQ ID NO: 25, provide that the sequenceidentity is not 100%, and a nucleotide sequence encoding iii) apolypeptide comprising an amino acid sequence set forth in SEQ ID NO: 26or iv) a polypeptide comprising an amino acid sequence which has atleast about 70%, such as at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 93%, at least 95%,at least 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 26, providethat the sequence identity is not 100%. Preferably, the polypeptidesassemble to form a protein having ATP sulfurylase activity.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such as avector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 25 and anucleotide sequence encoding a polypeptide comprising an amino acidsequence set forth in SEQ ID NO: 26.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such asvector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence which has at least about 70%, such asat least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 93%, at least 95%, at least 96%, at least 97%,at least 98%, or at least 99%, sequence identity to the amino acidsequence set forth in SEQ ID NO: 25, provide that the sequence identityis not 100%, and a nucleotide sequence encoding a polypeptide comprisingan amino acid sequence which has at least about 70%, such as at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 93%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99%, sequence identity to the amino acid sequence setforth in SEQ ID NO: 26, provide that the sequence identity is not 100%.Preferably, the polypeptides assemble to form a protein having ATPsulfurylase activity.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such asvector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence which has at least about 85%, such asat least about 90%, at least about 93%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, sequence identity to the aminoacid sequence set forth in SEQ ID NO: 25, provide that the sequenceidentity is not 100%, and a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence which has at least about 85%, such asat least about 90%, at least about 93%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, sequence identity to the aminoacid sequence set forth in SEQ ID NO: 26, provide that the sequenceidentity is not 100%. Preferably, the polypeptides assemble to form aprotein having ATP sulfurylase activity.

An alternative ATP sulfurylase encoding gene for use according to theinvention may for instance be the MET3 gene from Saccharomycescerevisiae (encoding SEQ ID NO: 68).

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such as avector) comprising a nucleotide sequence encoding i) a polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 68 or ii) apolypeptide comprising an amino acid sequence which has at least about70%, such as at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 93%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, sequence identity to the aminoacid sequence set forth in SEQ ID NO: 68. Preferably, the polypeptideaccording to ii) has ATP sulfurylase activity.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such as avector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 68.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such asvector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence which has at least about 70%, such asat least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 93%, at least 95%, at least 96%, at least 97%,at least 98%, or at least 99%, sequence identity to the amino acidsequence set forth in SEQ ID NO: 68. Preferably, the polypeptide has ATPsulfurylase activity.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such asvector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence which has at least about 85%, such asat least about 90%, at least about 93%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, sequence identity to the aminoacid sequence set forth in SEQ ID NO: 68. Preferably, the polypeptidehas ATP sulfurylase activity.

An alternative ATP sulfurylase encoding gene for use according to theinvention may for instance be the ATP sulfurylase encoding gene fromBacillus subtilis (encoding SEQ ID NO: 73).

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such as avector) comprising a nucleotide sequence encoding i) a polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 73 or ii) apolypeptide comprising an amino acid sequence which has at least about70%, such as at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 93%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, sequence identity to the aminoacid sequence set forth in SEQ ID NO: 73. Preferably, the polypeptideaccording to ii) has ATP sulfurylase activity.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such as avector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 73.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such asvector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence which has at least about 70%, such asat least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 93%, at least 95%, at least 96%, at least 97%,at least 98%, or at least 99%, sequence identity to the amino acidsequence set forth in SEQ ID NO: 73. Preferably, the polypeptide has ATPsulfurylase activity.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such asvector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence which has at least about 85%, such asat least about 90%, at least about 93%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, sequence identity to the aminoacid sequence set forth in SEQ ID NO: 73. Preferably, the polypeptidehas ATP sulfurylase activity.

In order to facilitate expression of the polypeptides in the host cell,the exogenous nucleic acid molecule may comprise suitable regulatoryelements such as a promoter that is functional in the host cell to causethe production of an mRNA molecule and that is operably linked to thenucleotide sequences encoding said polypeptides.

An APS kinase encoding gene for use according to the invention may forinstance be the cysC gene from Escherichia coli (encoding SEQ ID NO:27).

In certain instances a single polypeptide has been shown to possess bothan ATP sulfurylase and a 5′-adenylylsulfate kinase activity. Forexample, an ATP sulfurylase/APS kinase encoding gene has been isolatedfrom mouse (GenBank Accession No. U34883, Li et al. (1995) J BiolChem)70: 1945), and human (GenBank Accession No. AF033026, Yanagisawa(1998) Biosci Biotechnol Biochem 62: 1037) sources. Other examples ofsuch bifunctional enzyme include 3′-phosphoadenosine 5′-phosphosulfatesynthase enzymes (PAPSS) from rat (Rattus norvegicus) (SEQ ID NO: 71 or72).

According to certain embodiments, a recombinant host cell according tothe invention comprises an exogenous nucleic acid molecule (such as avector) comprising a nucleotide sequence encoding an APS kinase.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such as avector) comprising a nucleotide sequence encoding i) a polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 27 or ii) apolypeptide comprising an amino acid sequence which has at least about70%, such as at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 93%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, sequence identity to the aminoacid sequence set forth in SEQ ID NO: 27, provide that the sequenceidentity is not 100%. Preferably, said polypeptide according to ii) hasAPS kinase activity.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such as avector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 27.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such asvector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence which has at least about 70%, such asat least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 93%, at least 95%, at least 96%, at least 97%,at least 98%, or at least 99%, sequence identity to the amino acidsequence set forth in SEQ ID NO: 27, provide that the sequence identityis not 100%. Preferably, said polypeptide has APS kinase activity.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such asvector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence which has at least about 85%, such asat least about 90%, at least about 93%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, sequence identity to the aminoacid sequence set forth in SEQ ID NO: 27, provide that the sequenceidentity is not 100%. Preferably, said polypeptide has APS kinaseactivity.

An alternative APS kinase encoding gene for use according to theinvention may for instance be the MET14 gene from Saccharomycescerevisiae (encoding SEQ ID NO: 69).

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such as avector) comprising a nucleotide sequence encoding i) a polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 69 or ii) apolypeptide comprising an amino acid sequence which has at least about70%, such as at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 93%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, sequence identity to the aminoacid sequence set forth in SEQ ID NO: 69. Preferably, said polypeptideaccording to ii) has APS kinase activity.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such as avector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 69.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such asvector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence which has at least about 70%, such asat least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 93%, at least 95%, at least 96%, at least 97%,at least 98%, or at least 99%, sequence identity to the amino acidsequence set forth in SEQ ID NO: 69. Preferably, said polypeptide hasAPS kinase activity.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such asvector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence which has at least about 85%, such asat least about 90%, at least about 93%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, sequence identity to the aminoacid sequence set forth in SEQ ID NO: 69. Preferably, said polypeptidehas APS kinase activity.

An alternative APS kinase encoding gene for use according to theinvention may for instance be the APS kinase encoding gene from Bacillussubtilis (encoding SEQ ID NO: 74).

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such as avector) comprising a nucleotide sequence encoding i) a polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 74 or ii) apolypeptide comprising an amino acid sequence which has at least about70%, such as at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 93%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, sequence identity to the aminoacid sequence set forth in SEQ ID NO: 74. Preferably, said polypeptideaccording to ii) has APS kinase activity.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such as avector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 74.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such asvector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence which has at least about 70%, such asat least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 93%, at least 95%, at least 96%, at least 97%,at least 98%, or at least 99%, sequence identity to the amino acidsequence set forth in SEQ ID NO: 74. Preferably, said polypeptide hasAPS kinase activity.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such asvector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence which has at least about 85%, such asat least about 90%, at least about 93%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, sequence identity to the aminoacid sequence set forth in SEQ ID NO: 74. Preferably, said polypeptidehas APS kinase activity.

Alternatively, a polypeptide having both an ATP sulfurylase and a APSkinase activity can be used, such as a 3′-phosphoadenosine5′-phosphosulfate synthase (PAPSS).

According to certain embodiments, a recombinant host cell according tothe invention comprises an exogenous nucleic acid molecule (such as avector) comprising a nucleotide sequence encoding an 3′-phosphoadenosine5′-phosphosulfate synthase.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such as avector) comprising a nucleotide sequence encoding i) a polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 71 or ii) apolypeptide comprising an amino acid sequence which has at least about70%, such as at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 93%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, sequence identity to the aminoacid sequence set forth in SEQ ID NO: 71. Preferably, said polypeptideaccording to ii) has both an ATP sulfurylase and a APS kinase activity.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such as avector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 71.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such asvector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence which has at least about 70%, such asat least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 93%, at least 95%, at least 96%, at least 97%,at least 98%, or at least 99%, sequence identity to the amino acidsequence set forth in SEQ ID NO: 71. Preferably, said polypeptide hasboth an ATP sulfurylase and a APS kinase activity.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such asvector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence which has at least about 85%, such asat least about 90%, at least about 93%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, sequence identity to the aminoacid sequence set forth in SEQ ID NO: 71. Preferably, said polypeptidehas both an ATP sulfurylase and a APS kinase activity.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such as avector) comprising a nucleotide sequence encoding i) a polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 72 or ii) apolypeptide comprising an amino acid sequence which has at least about70%, such as at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 93%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, sequence identity to the aminoacid sequence set forth in SEQ ID NO: 72. Preferably, said polypeptideaccording to ii) has both an ATP sulfurylase and a APS kinase activity.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such as avector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 72.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such asvector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence which has at least about 70%, such asat least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 93%, at least 95%, at least 96%, at least 97%,at least 98%, or at least 99%, sequence identity to the amino acidsequence set forth in SEQ ID NO: 72. Preferably, said polypeptide hasboth an ATP sulfurylase and APS kinase activity.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such asvector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence which has at least about 85%, such asat least about 90%, at least about 93%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, sequence identity to the aminoacid sequence set forth in SEQ ID NO: 72. Preferably, said polypeptidehas both an ATP sulfurylase and a APS kinase activity.

In order to facilitate expression of the polypeptide in the host cell,the exogenous nucleic acid molecule may comprise suitable regulatoryelements such as a promoter that is functional in the host cell to causethe production of an mRNA molecule and that is operably linked to thenucleotide sequence encoding said polypeptide.

An PAP phosphatase encoding gene for use according to the invention mayfor instance be the cysQ gene from Escherichia coli (encoding SEQ ID NO:28).

According to certain embodiments, a recombinant host cell according tothe invention comprises an exogenous nucleic acid molecule (such as avector) comprising a nucleotide sequence encoding an PAP phosphatase.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such as avector) comprising a nucleotide sequence encoding i) a polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 28 or ii) apolypeptide comprising an amino acid sequence which has at least about70%, such as at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 93%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, sequence identity to the aminoacid sequence set forth in SEQ ID NO: 28, provide that the sequenceidentity is not 100%. Preferably, said polypeptide according to ii) hasPAP phosphatase activity.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such as avector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 28.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such asvector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence which has at least about 70%, such asat least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 93%, at least 95%, at least 96%, at least 97%,at least 98%, or at least 99%, sequence identity to the amino acidsequence set forth in SEQ ID NO: 28, provide that the sequence identityis not 100%. Preferably, said polypeptide has PAP phosphatase activity.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such asvector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence which has at least about 85%, such asat least about 90%, at least about 93%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, sequence identity to the aminoacid sequence set forth in SEQ ID NO: 28, provide that the sequenceidentity is not 100%. Preferably, said polypeptide has PAP phosphataseactivity.

An alternative PAP phosphatase encoding gene for use according to theinvention may for instance be the MET22 gene from Saccharomycescerevisiae (encoding SEQ ID NO: 70).

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such as avector) comprising a nucleotide sequence encoding i) a polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 70 or ii) apolypeptide comprising an amino acid sequence which has at least about70%, such as at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 93%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, sequence identity to the aminoacid sequence set forth in SEQ ID NO: 70. Preferably, said polypeptideaccording to ii) has PAP phosphatase activity.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such as avector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 70.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such asvector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence which has at least about 70%, such asat least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 93%, at least 95%, at least 96%, at least 97%,at least 98%, or at least 99%, sequence identity to the amino acidsequence set forth in SEQ ID NO: 70. Preferably, said polypeptide hasPAP phosphatase activity.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such asvector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence which has at least about 85%, such asat least about 90%, at least about 93%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, sequence identity to the aminoacid sequence set forth in SEQ ID NO: 70. Preferably, said polypeptidehas PAP phosphatase activity.

An alternative PAP phosphatase encoding gene for use according to theinvention may for instance be the PAP phosphatase encoding gene fromBacillus subtilits (encoding SEQ ID NO: 75).

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such as avector) comprising a nucleotide sequence encoding i) a polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 75 or ii) apolypeptide comprising an amino acid sequence which has at least about70%, such as at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 93%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, sequence identity to the aminoacid sequence set forth in SEQ ID NO: 75. Preferably, said polypeptideaccording to ii) has PAP phosphatase activity.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such as avector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 75.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such asvector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence which has at least about 70%, such asat least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 93%, at least 95%, at least 96%, at least 97%,at least 98%, or at least 99%, sequence identity to the amino acidsequence set forth in SEQ ID NO: 75. Preferably, said polypeptide hasPAP phosphatase activity.

According to particular embodiments, a recombinant host cell accordingto the invention comprises an exogenous nucleic acid molecule (such asvector) comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence which has at least about 85%, such asat least about 90%, at least about 93%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, sequence identity to the aminoacid sequence set forth in SEQ ID NO: 75. Preferably, said polypeptidehas PAP phosphatase activity.

In order to facilitate expression of the polypeptide in the host cell,the exogenous nucleic acid molecule may comprise suitable regulatoryelements such as a promoter that is functional in the host cell to causethe production of an mRNA molecule and that is operably linked to thenucleotide sequence encoding said polypeptide.

It is understood that the details given herein with respect to arecombinant host cell apply to other aspects of the invention, inparticular to the processes and uses according to the invention, whichare described in more detail below.

Methods and Uses

The present invention provides processes for the production of sulfatedphenolic compounds. Particularly, a process for the production of asulfated phenolic compound is provided comprising:

(i′) contacting a medium comprising a phenolic compound with a firstrecombinant host cell; wherein the first recombinant host cell comprisesa heterologous polypeptide having an aryl sulfotransferase activity; or

(i″) contacting a medium comprising a fermentable carbon substrate witha first recombinant host cell; wherein the first recombinant host cellcomprises a heterologous polypeptide having an aryl sulfotransferaseactivity; or

(i′″) contacting a medium comprising a precursor of a phenolic compoundwith a first recombinant host cell; wherein the first recombinant hostcell comprises a heterologous polypeptide having an arylsulfotransferase activity.

According to certain embodiments, the process for the production of asulfated phenolic compound comprises:

(i′) contacting a medium comprising a phenolic compound with a firstrecombinant host cell; wherein the first recombinant host cell comprisesa heterologous polypeptide having an aryl sulfotransferase activity.

According to other certain embodiments, the process for the productionof a sulfated phenolic compound comprises:

(i″) contacting a medium comprising a fermentable carbon substrate witha first recombinant host cell; wherein the first recombinant host cellcomprises a heterologous polypeptide having an aryl sulfotransferaseactivity.

According to other certain embodiments, the process for the productionof a sulfated phenolic compound comprises:

(i′″) contacting a medium comprising a precursor of a phenolic compoundwith a first recombinant host cell; wherein the first recombinant hostcell comprises a heterologous polypeptide having an arylsulfotransferase activity.

The medium employed may be any conventional medium suitable forculturing the host cell in question, and may be composed according tothe principles of the prior art. The medium will usually contain allnutrients necessary for the growth and survival of the respective hostcell, such as carbon and nitrogen sources and other inorganic salts.Suitable media, e.g. minimal or complex media, are available fromcommercial suppliers, or may be prepared according to publishedreceipts, e.g. the American Type Culture Collection (ATCC) Catalogue ofstrains. Non-limiting standard medium well known to the skilled personinclude Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth, MSbroth, Yeast Peptone Dextrose, BMMY, GMMY, or Yeast Malt Extract (YM)broth, which are all commercially available. A non-limiting example ofsuitable media for culturing bacterial cells, such as B. subtilis, L.lactis or E. coli cells, including minimal media and rich media such asLuria Broth (LB), M9 media, M17 media, SA media, MOPS media, TerrificBroth, YT and others. Suitable media for culturing eukaryotic cells,such as yeast cells, are RPMI 1640, MEM, DMEM, all of which may besupplemented with serum and/or growth factors as required by theparticular host cell being cultured. The medium for culturing eukaryoticcells may also be any kind of minimal media such as Yeast minimal media.

The fermentable carbon substrate may be any suitable carbon substrateknow in the art, and in particularly any carbon substrate commonly usedin the cultivation of microorganisms and/or fermentation. Non-limitingexamples of suitable fermentable carbon substrates include carbohydrates(e.g., C5 sugars such as arabinose or xylose, or C6 sugars such asglucose), glycerol, glycerine, acetate, dihydroxyacetone, one-carbonsource, methanol, methane, oils, animal fats, animal oils, plant oils,fatty acids, lipids, phospholipids, glycerolipids, monoglycerides,diglycerides, triglycerides, renewable carbon sources, polypeptides(e.g., a microbial or plant protein or peptide), yeast extract,component from a yeast extract, peptone, casaminoacids or anycombination of two or more of the foregoing.

According to certain embodiments, the carbon substrate is selected fromthe group consisting of C5 sugars (such as arabinose or xylose), C6sugars (such as glucose or fructose), lactose, sucrose, glycerol,glycerine, acetate, Corn steep liquor, yeast extract, component from ayeast extract, peptone, casaminoacids or combinations thereof.

According to certain embodiments, the medium comprises glucose.

According to certain other embodiments, the medium comprises glycerol.

According to certain other embodiments, the medium comprises acetate.

It is also contemplated to use starch as a carbon substrate. Dependingon the microorganism used, the metabolization of starch may require thesupplementation of beta-glucosidase, such as the beta-glucosidase fromNeurospora crassa, to the medium. Alternatively, a recombination hostcell according to the invention may be further genetically modified toexpress a beta-glucosidase, such as the beta-glucosidase from Neurosporacrassa.

When a fermentable carbon substrate is employed it is thus possible thatthe recombinant host cell produces the phenolic compound or a precursorthereof directly from such primary carbon substrate.

According to certain embodiments, the process further comprises:

(ii) culturing the first recombinant host cell under suitable conditionsfor the production of the corresponding sulfated phenolic compound.

Suitable conditions for culturing the respective host cell are wellknown to the skilled person. Typically, the recombinant host cell iscultured at a temperature ranging from about 23 to about 60° C., such asfrom about 25 to about 40° C., such as at about 37° C. The pH of themedium may range from pH 1.0 to pH 14.0, such as from about pH 1 toabout pH 2, from about pH 4 to about pH 11, from about pH 5 to about pH10, from about pH 6 to about pH 10, or from about pH 7 to about pH 9.5,e.g. at pH 6.0, pH 7.0, pH. 7.5, pH 8.0, pH 8.5, pH 9.0, pH 9.5, pH10.0, pH 10.5 or pH 11.0.

The process may further comprise iii) recovering the sulfated phenoliccompound. The sulfated phenolic compound may be recovered byconventional method for isolation and purification chemical compoundsfrom a medium. Well-known purification procedures include centrifugationor filtration, precipitation, and chromatographic methods such as e.g.ion exchange chromatography, gel filtration chromatography, etc.

Further provided is a process for the production of a sulfated phenoliccompound, such as zosteric acid, the method comprises sulfating aphenolic compound, such as p-coumaric acid, using a polypeptide havingaryl sulfotransferase activity as detailed herein. Such polypeptide maybe selected from the group consisting of:

a) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1);

b) a polypeptide comprising an amino acid sequence which has at leastabout 70%, such as at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 93%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1); or

c) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1),wherein 1 to 50, such as 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1to 15, 1 to 10, 1 to 5, 1 to 3, amino acid residues are substituted,deleted and/or inserted.

Suitable conditions for the sulfation reaction are well known to theskilled person. Typically, the sulfation reaction takes place at atemperature ranging from about 23 to about 60° C., such as from about 25to about 40° C., such as at about 37° C. The deamination reaction maytake place at a pH ranging from pH 1.0 to pH 14.0, such as from about pH2 to about pH 11, such as from about pH 5 to about pH 10, from about pH6 to about pH 10, or from about pH 7 to about pH 9.5, e.g. at pH 6.0, pH7.0, pH. 7.5, pH 8.0, pH 8.5, pH 9.0, pH 9.5, pH 10.0, pH 10.5 or pH11.0.

Also provide is the use of a polypeptide in the sulfation of a phenoliccompound, said polypeptide having aryl sulfotransferase activity asdetailed herein. Such polypeptide may be selected from the groupconsisting of:

a) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1);

b) a polypeptide comprising an amino acid sequence which has at leastabout 70%, such as at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 93%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1); or

c) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1),wherein 1 to 50, such as 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1to 15, 1 to 10, 1 to 5, 1 to 3, amino acid residues are substituted,deleted and/or inserted.

For the purpose of this specification and the appended claims, it shouldbe understood that the phenolic compounds include those compounds inwhich a hydroxyl group is directly attached to a benzenoid carbon atom,and which compounds may or may not contain other substituent groups.

According to certain embodiments, the phenolic compound is a compoundrepresented by the general formula (I):

wherein at least one of R₁, R₂, R₃, R₄, and R₅ being an hydroxyl group(—OH);

wherein R₁, R₂, R₃, R₄, R₅ and R₆ are independently selected from thegroup consisting of halide, hydrogen, hydroxyl (—OH), —OR₇, —OCOR₇,—NR₇R₈, —COR₇, —COOR₇, —SR₇, —OSO₃R₇, —OCSR₇, —POR₇R₈, alkyl, alkenyl,alkynyl, aryl, and heteroaryl; wherein R₇, and R₈ are independentlyselected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl,aryl, and heteroaryl;

wherein R₁, R₂, R₃, R₄, R₅ and R₆, are optionally linked with a bridgemember Y_(n), thereby forming one or more rings, Y_(n) being a bond or aC₁₋₁₂ alkyl or an aryl, a carbocyclic, a heterocyclic or aheteroaromatic structure having 1-3 rings, 3-8 ring members in each and0 to 4 heteroatoms, or a heteroalkyl comprising 1 to 12 heteroatomsselected from the group consisting of N, O, S, S(O)₁₋₂ and carbonyl, andwherein n is an integer between 1 and 12.

Specific examples of compounds of Formula I include, but are not limitedto, reservatrol, o-, m-, and p-coumaric acid, caffeic acid, ferulicacid, sinapic acid, curcumin, rosmarinic acid, sinapyl alcohol,coniferyl alcohol, and salvianolic acid.

A precursor of a phenolic compound according to Formula I may be acompound represented by the general Formula (p-I):

wherein at least one of R₁, R₂, R₃, R₄, and R₅ being an hydroxyl group(—OH);

wherein R₁, R₂, R₃, R₄, R₅ and R₆ are independently selected from thegroup consisting of halide, hydrogen, hydroxyl (—OH), —OR₇, —OCOR₇,—NR₇R₈, —COR₇, —COOR₇, —SR₇, —OSO₃R₇, —OCSR₇, —POR₇R₈, alkyl, alkenyl,alkynyl, aryl, and heteroaryl; wherein R₇, and R₈ are independentlyselected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl,aryl, and heteroaryl;

wherein R₁, R₂, R₃, R₄, R₅ and R₆, are optionally linked with a bridgemember Y_(n), thereby forming one or more rings, Y_(n) being a bond or aC₁₋₁₂ alkyl or an aryl, a carbocyclic, a heterocyclic or aheteroaromatic structure having 1-3 rings, 3-8 ring members in each and0 to 4 heteroatoms, or a heteroalkyl comprising 1 to 12 heteroatomsselected from the group consisting of N, O, S, S(O)₁₋₂ and carbonyl, andwherein n is an integer between 1 and 12.

Such a precursor may be converted to the phenolic compound by arecombinant host cell according to the invention, comprising apolypeptide having tyrosine ammonia lyase activity. Such polypeptidewill eliminate ammonia from the precursor of Formula (p-I) under theformation of the corresponding molecule of Formula I. Preferably, thep-I precursor is the L-isomer.

According to certain embodiments, the precursor of a phenolic compoundas employed in step (i′″) is a compound of the general Formula (p-I) asdefined herein.

According to certain other embodiments, the phenolic compound is acompound represented by the general formula (II):

wherein at least one of R₁, R₂, R₃, R₄, and R₅ being an hydroxyl group(—OH);

wherein R₁, R₂, R₃, R₄, R₅ and R₆ are independently selected from thegroup consisting of halide, hydrogen, hydroxyl (—OH), —OR₇, —OCOR₇,—NR₇R₈, —COR₇, —COOR₇, —SR₇, —OSO₃R₇, —OCSR₇, —POR₇R₈, alkyl, alkenyl,alkynyl, aryl, and heteroaryl; wherein R₇, and R₈ are independentlyselected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl,aryl, and heteroaryl;

wherein R₁, R₂, R₃, R₄, R₅ and R₆, are optionally linked with a bridgemember Y_(n), thereby forming one or more rings, Y_(n) being a bond or aC₁₋₁₂ alkyl or an aryl, a carbocyclic, a heterocyclic or aheteroaromatic structure having 1-3 rings, 3-8 ring members in each and0 to 4 heteroatoms, or a heteroalkyl comprising 1 to 12 heteroatomsselected from the group consisting of N, O, S, S(O)₁₋₂ and carbonyl, andwherein n is an integer between 1 and 12.

According to certain embodiments, R₆ is —COOR₇.

According to certain embodiments, R₇ is hydrogen.

According to certain embodiments, R₂ is hydroxyl (—OH).

According to certain embodiments, R₃ is hydroxyl (—OH).

According to certain embodiments, R₄ is hydroxyl (—OH).

According to certain embodiments, each of R₁, R₂, R₄ and R₅ is hydrogen.

According to certain embodiments, each of R₁, R₂, and R₅ is hydrogen.

According to particular embodiments, the phenolic compound is p-coumaricacid (Formula I: R₁=H, R₂=H, R₃=OH, R₄=H, R₅=H, R₆=COOH).

According to other particular embodiments, the phenolic compound iscaffeic acid (Formula I: R₁=H, R₂=H, R₃=OH, R₄=OH, R₅=H, R₆=COOH).

According to other particular embodiments, the phenolic acid is ferulicacid (Formula I: R₁=H, R₂=OCH₃, R₃=OH, R₄=H, R₅=H, R₆=COOH).

According to other particular embodiments, the phenolic acid is sinapicacid (Formula I: R₁=H, R₂=OCH₃, R₃=OH, R₄=OCH₃, R₅=H, R₆=COOH).

According to other particular embodiments, the phenolic compound isresveratrol (Formula I: R₁=H, R₂=OH, R₃=H, R₄=OH, R₅=H,R₆=p-hydroxyphenyl).

According to other particular embodiments, the phenolic compound isvanillin (Formula II: R₁=H, R₂=H, R₃=OH, R₄=OCH₃, R₅=H, R₆=H).

According to certain embodiments, the phenolic compound is ahydroxycinnamic acid.

According to certain embodiments, the phenolic compound is a compoundrepresented by the general formula (I), wherein R₁ is hydrogen; R₂, R₃and R₄ independently are selected from the group consisting of hydrogen(H), hydroxyl (—OH), C₁₋₆-alkyl and C₁₋₆-Alkoxy, provided that at leastone of R₂, R₃ and R₄ is hydroxyl (—OH); R₅ is hydrogen, and R₆ is COOH.

According to certain embodiments, the precursor of a phenolic compoundas employed in step (i′″) is a compound of the general Formula (p-I),wherein R₁ is hydrogen; R₂, R₃ and R₄ independently are selected fromthe group consisting of hydrogen (H), hydroxyl (—OH), C₁₋₆-alkyl andC₁₋₆-Alkoxy, provided that at least one of R₂, R₃ and R₄ is hydroxyl(—OH); R₅ is hydrogen, and R₆ is COOH.

According to certain embodiment, the sulfated phenolic compound obtainedin according to the present invention is zosteric acid.

Suitable sulfate donor molecules metabolized by a polypeptide havingaryl sulfotransferase activity are well-known to one skilled in the art.Non-limiting examples include 3′-phosphoadenosine 5′-phosphosulfate(PAPS), para-nitrophenyl sulfate (pNPS) and 4-methylumbelliferyl sulfate(MUS). Such sulfate donor molecules may be employed to facilitate thesulfation of phenolic compounds in accordance with the invention.

The medium employed for culturing the recombinant host cell may be anyconventional medium suitable for culturing the host cell in question,and may be composed according to the principles of the prior art. Themedium will usually contain all nutrients necessary for the growth andsurvival of the respective host cell, such as carbon and nitrogensources and other inorganic salts, such as sulfate salts. Suitablemedia, e.g. minimal or complex media, are available from commercialsuppliers, or may be prepared according to published receipts, e.g. theAmerican Type Culture Collection (ATCC) Catalogue of strains.Non-limiting standard medium well known to the skilled person includeLuria Bertani (LB) broth, Sabouraud Dextrose (SD) broth, MS broth, YeastPeptone Dextrose, BMMY, GMMY, or Yeast Malt Extract (YM) broth, whichare all commercially available. A non-limiting example of suitable mediafor culturing bacterial cells, such as B. subtilis, L. lactis or E. colicells, including minimal media and rich media such as Luria Broth (LB),M9 media, M17 media, SA media, MOPS media, Terrific Broth, YT andothers. Suitable media for culturing eukaryotic cells, such as yeastcells, are RPMI 1640, MEM, DMEM, all of which may be supplemented withserum and/or growth factors as required by the particular host cellbeing cultured. The medium for culturing eukaryotic cells may also beany kind of minimal media such as Yeast minimal media.

Certain Definitions

“Aryl sulfotransferase activity” as used herein refers to the ability ofa polypeptide to catalyze the catalyze the transfer of a sulfate groupfrom a donor molecule to an aryl acceptor molecule.

“Tyrosine ammonia lyase activity” as used herein refers to the abilityof a polypeptide to catalysed the conversion of L-tyrosine intop-coumaric acid.

“Phenylalanine ammonia lyase activity” as used herein refers to theability of a polypeptide to catalysed the conversion of L-phenylalanineinto trans-cinnamic acid.

“ATP sulfurylase” as used herein refers to an enzyme that catalyzes thereaction: ATP+sulfate=diphosphate+adenosine 5′-phosphosulfate (APS).

“APS kinase” as used herein refers to an enzyme that catalyzes thereaction: ATP+adenosine 5′-phosphosulfate (APS)=ADP+3′-phosphoadenosine5′-phosphosulfate (PAPS).

“PAP phosphatase” as used herein refers to an enzyme that catalyzes thereaction: 3′-phosphoadenosine 5′-phosphate (PAP)+H₂O=AMP+phosphate.

“Polypeptide,” or “protein” are used interchangeably herein to denote apolymer of at least two amino acids covalently linked by an amide bond,regardless of length or post-translational modification (e.g.,glycosylation, phosphorylation, lipidation, myristilation,ubiquitination, etc.). Included within this definition are D- andL-amino acids, and mixtures of D- and L-amino acids.

“Nucleic acid” or “polynucleotide” are used interchangeably herein todenote a polymer of at least two nucleic acid monomer units or bases(e.g., adenine, cytosine, guanine, thymine) covalently linked by aphosphodiester bond, regardless of length or base modification.

“Recombinant” or “non-naturally occurring” when used with reference to,e.g., a host cell, nucleic acid, or polypeptide, refers to a material,or a material corresponding to the natural or native form of thematerial, that has been modified in a manner that would not otherwiseexist in nature, or is identical thereto but produced or derived fromsynthetic materials and/or by manipulation using recombinant techniques.Non-limiting examples include, among others, recombinant host cellsexpressing genes that are not found within the native (non-recombinant)form of the cell or express native genes that are otherwise expressed ata different level.

“Substitution” or “substituted” refers to modification of thepolypeptide by replacing one amino acid residue with another, forinstance the replacement of an Arginine residue with a Glutamine residuein a polypeptide sequence is an amino acid substitution.

“Conservative substitution” refers to a substitution of an amino acidresidue with a different residue having a similar side chain, and thustypically involves substitution of the amino acid in the polypeptidewith amino acids within the same or similar class of amino acids. By wayof example and not limitation, an amino acid with an aliphatic sidechain may be substituted with another aliphatic amino acid, e.g.,alanine, valine, leucine, and isoleucine; an amino acid with hydroxylside chain is substituted with another amino acid with a hydroxyl sidechain, e.g., serine and threonine; an amino acid having an aromatic sidechain is substituted with another amino acid having an aromatic sidechain, e.g., phenylalanine, tyrosine, tryptophan, and histidine; anamino acid with a basic side chain is substituted with another aminoacid with a basic side chain, e.g., lysine and arginine; an amino acidwith an acidic side chain is substituted with another amino acid with anacidic side chain, e.g., aspartic acid or glutamic acid; and ahydrophobic or hydrophilic amino acid is replaced with anotherhydrophobic or hydrophilic amino acid, respectively.

“Non-conservative substitution” refers to substitution of an amino acidin a polypeptide with an amino acid with significantly differing sidechain properties. Non-conservative substitutions may use amino acidsbetween, rather than within, the defined groups and affects (a) thestructure of the peptide backbone in the area of the substitution (e.g.,proline for glycine) (b) the charge or hydrophobicity, or (c) the bulkof the side chain. By way of example and not limitation, an exemplarynon-conservative substitution can be an acidic amino acid substitutedwith a basic or aliphatic amino acid; an aromatic amino acid substitutedwith a small amino acid; and a hydrophilic amino acid substituted with ahydrophobic amino acid.

“Deletion” or “deleted” refers to modification of the polypeptide byremoval of one or more amino acids in the reference polypeptide.Deletions can comprise removal of 1 or more amino acids, 2 or more aminoacids, 5 or more amino acids, 10 or more amino acids, 15 or more aminoacids, or 20 or more amino acids, up to 10% of the total number of aminoacids, or up to 20% of the total number of amino acids making up thepolypeptide while retaining enzymatic activity and/or retaining theimproved properties of an engineered enzyme. Deletions can be directedto the internal portions and/or terminal portions of the polypeptide, invarious embodiments, the deletion can comprise a continuous segment orcan be discontinuous.

“Insertion” or “inserted” refers to modification of the polypeptide byaddition of one or more amino acids to the reference polypeptide.Insertions can comprise addition of 1 or more amino acids, 2 or moreamino acids, 5 or more amino acids, 10 or more amino acids, 15 or moreamino acids, or 20 or more amino acids. Insertions can be in theinternal portions of the polypeptide, or to the carboxy or aminoterminus. The insertion can be a contiguous segment of amino acids orseparated by one or more of the amino acids in the referencepolypeptide.

“Host cell” as used herein refers to a living cell or microorganism thatis capable of reproducing its genetic material and along with itrecombinant genetic material that has been introduced into it—e.g., viaheterologous transformation.

“Expression” includes any step involved in the production of apolypeptide (e.g., encoded enzyme) including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

As used herein, “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid molecule to which it has been linked.One type of vector is a “plasmid”, which refers to a circular doublestranded nucleic acid loop into which additional nucleic acid segmentscan be ligated. Certain vectors are capable of directing the expressionof genes to which they are operatively linked. Such vectors are referredto herein as “expression vectors”. Certain other vectors are capable offacilitating the insertion of a exogenous nucleic acid molecule into agenome of a host cell. Such vectors are referred to herein as“transformation vectors”. In general, vectors of utility in recombinantnucleic acid techniques are often in the form of plasmids. In thepresent specification, “plasmid” and “vector” can be usedinterchangeably as the plasmid is the most commonly used form of avector. Large numbers of suitable vectors are known to those of skill inthe art and commercially available.

As used herein, “promoter” refers to a sequence of DNA, usually upstream(5′) of the coding region of a structural gene, which controls theexpression of the coding region by providing recognition and bindingsites for RNA polymerase and other factors which may be required forinitiation of transcription. The selection of the promoter will dependupon the nucleic acid sequence of interest. A “promoter functional in ahost cell” refers to a “promoter” which is capable of supporting theinitiation of transcription in said cell, causing the production of anmRNA molecule.

As used herein, “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequence. A promoter sequence is “operably-linked” to a gene when it isin sufficient proximity to the transcription start site of a gene toregulate transcription of the gene.

“Percentage of sequence identity,” “% sequence identity” and “percentidentity” are used herein to refer to comparisons between an amino acidsequence and a reference amino acid sequence. The “% sequence identify”,as used herein, is calculated from the two amino acid sequences asfollows: The sequences are aligned using Version 9 of the GeneticComputing Group's GAP (global alignment program), using the defaultBLOSUM62 matrix (see below) with a gap open penalty of −12 (for thefirst null of a gap) and a gap extension penalty of −4 (for eachadditional null in the gap). After alignment, percentage identity iscalculated by expressing the number of matches as a percentage of thenumber of amino acids in the reference amino acid sequence.

The following BLOSUM62 matrix is used:

Ala 4 Arg −1 5 Asn −2 0 6 Asp −2 −2 1 6 Cys 0 −3 −3 −3 9 Gln −1 1 0 0 −35 Glu −1 0 0 2 −4 2 5 Gly 0 −2 0 −1 −3 −2 −2 6 His −2 0 1 −1 −3 0 0 −2 8Ile −1 −3 −3 −3 −1 −3 −3 −4 −3 4 Leu −1 −2 −3 −4 −1 −2 −3 −4 −3 2 4 Lys−1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 Met −1 −1 −2 −3 −1 0 −2 −3 −2 1 2 −1 5Phe −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 Pro −1 −2 −2 −1 −3 −1 −1 −2 −2−3 −3 −1 −2 −4 7 Ser 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1 −2 −1 4 Thr 0 −1 0−1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 Trp −3 −3 −4 −4 −2 −2 −3 −2 −2−3 −2 −3 −1 1 −4 −3 −2 11 Tyr −2 −2 −2 −3 −2 −1 −2 −3 2 −1 −1 −2 −1 3 −3−2 −2 2 7 Val 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0 −3 −1 4 AlaArg Asn Asp Cys Gln Glu Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp TyrVal

“Reference sequence” or “reference amino acid sequence” refers to adefined sequence to which another sequence is compared. In the contextof the present invention a reference amino acid sequence may be an aminoacid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23.

Aliphatic radicals/groups, as referred herein, are optionally mono- orpolysubstituted and may be branched or unbranched, saturated orunsaturated. Unsaturated aliphatic groups, as defined in herein, includealkyl, alkenyl and alkinyl radicals. Preferred aliphatic radicalsaccording to the present invention include but are not restricted tomethyl, ethyl, vinyl (ethenyl), ethinyl, propyl, n-propyl, isopropyl,allyl (2-propenyl), 1-propinyl, methylethyl, butyl, n-butyl, iso-butyl,sec-butyl, tert-butyl butenyl, butinyl, 1-methylpropyl, 2-methylpropyl,1,1-dimethylethyl, pentyl, n-pentyl, 1,1-dimethylpropyl,1,2-dimethylpropyl, 2,2-dimethylpropyl, hexyl, 1-methylpentyl, n-heptyl,n-octyl, n-nonyl and n-decyl. Preferred substituents for aliphaticradicals, according to the present invention, are a C₁₋₄ alkyl group, alinear or branched C₁₋₆ alkoxy group, F, Cl, I, Br, CF₃, CH₂F, CHF₂, CN,OH, SH, NH₂, oxo, (C═O)R′, SR′, SOR′, SO₂R′, NHR′, NR′R″ whereby R′ andoptionally R″ for each substitutent independently represents a linear orbranched C₁₋₆-alkyl group.

“Alkyl”, “alkyl radical” or group as used herein means saturated, linearor branched hydrocarbons, which can be unsubstituted or mono- orpolysubstituted. Thus, unsaturated alkyl is understood to encompassalkenyl and alkinyl groups, like e.g. —CH═CH—CH₃ or —C≡C—CH₃, whilesaturated alkyl encompasses e.g. —CH₃ and —CH₂—CH₃. “C₁₋₁₂-alkyl”includes C₁₋₂-alkyl, C₁₋₃-alkyl, C₁₋₄-alkyl, and C₁₋₆-alkyl, C₁₋₆-alkyl,C₁₋₇-alkyl, C₁₋₈-alkyl, C₁₋₃-alkyl, C₁₋₁₀-alkyl, and C₁₋₁₁-alkyl. Inthese radicals, C₁₋₂-alkyl represents C₁- or C₂-alkyl, C₁₋₃-alkylrepresents C₁-, C₂- or C₃-alkyl, C₁₋₄-alkyl represents C₁-, C₂-, C₃- orC₄-alkyl, C₁₋₆-alkyl represents C₁-, C₂-, C₃-, C₄-, or C₅-alkyl,C₁₋₆-alkyl represents C₁-, C₂-, C₃-, C₄-, C₅- or C₆-alkyl etc. The alkylradicals may be methyl, ethyl, vinyl (ethenyl), propyl, allyl(2-propenyl), 1-propinyl, methylethyl, butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, pentyl, 1,1-dimethylpropyl,1,2-dimethylpropyl, 2,2-dimethylpropyl, hexyl, 1-methylpentyl, ifsubstituted also CHF₂, CF₃ or CH₂OH etc. These alkyl, alkenyl or alkinylradicals may optionally be mono- or polysubstituted by substitutentsindependently selected from a C₁₋₄ alkyl group, a linear or branchedC₁₋₆ alkoxy group, F, Cl, I, Br, CF₃, CH₂F, CHF₂, CN, OH, SH, NH₂,(C═O)R′, SR′, SOR′, SO₂R′, NHR′, NR′R″ whereby R′ and optionally R″ foreach substitutent independently represents linear or branched C₁₋₆-alkylgroup.

“Aryl” or “aryl radical” as herein is understood as meaning ring systemswith at least one aromatic ring but without heteroatoms even in only oneof the rings. These aryl radicals may optionally be mono- orpolysubstituted by substitutents independently selected from a C₁₋₄alkyl group, a linear or branched C₁₋₆ alkoxy group, an optionally atleast mono-substituted phenyl group, F, Cl, I, Br, CF₃, CH₂F, CHF₂, CN,OH, SH, NH₂, oxo, (C═O)R′, SR′, SOR′, SO₂R′, N(C═O)—OR′, NHR′, NR′R″whereby R′ and optionally R″ for each substitutent independentlyrepresents a linear or branched C₁₋₆-alkyl group. Preferred examples ofaryl radicals include but are not restricted to phenyl, naphthyl,fluoranthenyl, fluorenyl, tetralinyl or indanyl or anthracenyl radicals,which may optionally be mono- or polysubstituted, if not definedotherwise.

“Alkyl-aryl” or “alkyl-aryl radical” as used herein comprises a linearor branched, optionally at least mono-substituted alkyl chain which isbonded to an aryl group, as defined above. A preferred alkyl-arylradical is a benzyl group, wherein the alkyl chain is optionallybranched or substituted. Preferred substituents for alkyl-aryl radicals,according to the present invention, are F, Cl, Br, I, NH₂, SH, OH, SO₂,CF₃, carboxy, amido, cyano, carbamyl, nitro, phenyl, benzyl, —SO₂NH₂,C₁₋₆ alkyl and/or C₁₋₆-alkoxy.

“Heteroaryl” or “heteroaryl radical” as used herein is understood asmeaning heterocyclic ring systems which have at least one aromatic ringand may optionally contain one or more heteroatoms from the groupconsisting of nitrogen, oxygen and/or sulfur and may optionally be mono-or polysubstituted by substitutents independently selected from a C₁₋₄alkyl group, a linear or branched C₁₋₆ alkoxy group, F, Cl, I, Br, CF₃,CH₂F, CHF₂, CN, OH, SH, NH₂, oxo, (C═O)R′, SR′, SOR′, SO₂R′, NHR′, NR′R″whereby R′ and optionally R″ for each substitutent independentlyrepresents a linear or branched C₁-6-alkyl group. Preferred examples ofheteroaryls include but are not restricted to furan, benzofuran,thiophene, benzothiophene, pyrrole, pyridine, pyrimidine, pyridazine,pyrazine, quinoline, isoquinoline, phthalazine, benzo-1,2,5-thiadiazole,benzothiazole, indole, benzotriazole, benzodioxolane, benzodioxane,benzimidzole, carbazole and quinazoline.

“Alkoxy”, “alkoxy radical” or group as used herein means an “alkyl”singular bonded to oxygen. “C₁₋₆-alkoxy” includes C₁₋₂-alkoxy,C₁₋₃-alkoxy, C₁₋₄-alkoxy, and C₁₋₆-alkoxy, as well as C₂₋₃-alkoxy,C₂₋₄-alkoxy, C₂₋₆-alkoxy, C₃₋₄-alkoxy, C₃₋₆-alkoxy, and C₄₋₆-alkoxy. Inthese radicals, C₁₋₂-alkoxy represents C1- or C2-alkoxy, C₁₋₃-alkoxyrepresents C₁-, C₂- or C₃-alkoxy, C₁₋₄-alkyl represents C₁-, C₂-, C₃- orC₄-alkoxy, C₁₋₆-alkoxy represents C₁-, C₂-, C₃-, C₄-, or C₅-alkoxy,C₁₋₆-alkoxy represents C₁-, C₂-, C₃-, C₄-, C₅- or C₆-alkoxy. The alkoxyradicals may be methoxy, ethoxy, propoxy, butoxy, pentyloxy or hexyloxy.

The term “precursor of a phenolic compound” refers to any compound thatmay be converted to a phenolic compound by a host cells as describedherein.

Where a numerical limit or range is stated herein, the endpoints areincluded. Also, all values and sub ranges within a numerical limit orrange are specifically included as if explicitly written out.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples, which areprovided herein for purposes of illustration only, and are not intendedto be limiting unless otherwise specified.

Examples Example 1—Production of Zosteric Acid in E. coli

A range of aryl sulfotransferases including SULT1A1 Rattus norvegicus(SEQ ID NO: 1), SULT1A1 Homo sapiens (SEQ ID NO: 2), SULT1A1 Equuscaballus (SEQ ID NO: 3), SULT1A1 Sus scrofa domesticus (SEQ ID NO: 4),SULT1A1 Canis lupus familiaris (SEQ ID NO: 5) and SULT1E1 Gallus gallusdomesticus (SEQ ID NO: 6) were expressed in Escherichia coli. Therespective genes encoding SEQ ID NO. 1, 3, 4, 5, and 6 were clonedamplified from liver tissue cDNA (Zyagen) by PCR using the primerslisted in Table 1. The nucleotide sequence of the gene encoding SEQ IDNO: 2 was codon optimized for expression in Escherichia coli (GeneArt,Life Technologies) and amplified by PCR using the primers in Table 1.The pETDuet-1 plasmid was digested with restriction endonucleases NcoIand SalI. The PCR products were then individually cloned into theplasmid pETDuet-1 using the Gibson reaction (New England Biolabs). Theresulting plasmids were transformed into BL21(DE3)pLysS (LifeTechnologies). FIG. 1 shows the plasmid map of the plasmid encodingSULT1A1 Rattus norvegicus (SEQ ID NO: 1).

TABLE 1 Overview of enzymes and primers for cloning arylsulfotransferases SEQ Rev ID NO Name Fwd Primer Primer 1 SULT1A1 Rattusnorvegicus CBJP472 CBJP473 2 SULT1A1 Homo sapiens CBJP470 CBJP471 3SULT1A1 Equus caballus CBJP499 CBJP500 4 SULT1A1 Sus scrofa domesticusCBJP505 CBJP506 5 SULT1A1 Canis lupus familiaris CBJP503 CBJP504 6SULT1E1 Gallus gallus domesticus CBJP501 CBJP502

The strains were grown in M9 minimal media containing glucose as acarbon source, and 0.1 mM IPTG for induction of gene expression as wellas 0.1 mM p-coumaric acid (pHCA). After four days of growth, sampleswere withdrawn by filtration and analyzed by HPLC.

The concentration of p-courmaric acid (pHCA) and zosteric acid in thesupernatant was quantified by high performance (HPLC) and compared tochemical standards. HPLC was done on a Thermo setup using a HS-F5 columnand mobile phases: 5 mM ammonium formate pH 4.0 (A) and acetonitrile (B)at 1.5 mL min-1, using a gradient elution starting at 5% B. From 0.5 minafter injection to 7 min, the fraction of B increased linearly from 5%to 60%, and between 9.5 min and 9.6 the fraction of B decreased back to5%, and remaining there until 12 min. pHCA and zosteric acid werequantified by measuring absorbance at 277 nm.

Table 2 shows the remaining pHCA and the produced zosteric acid in theculture media. Zosteric acid was formed with an aryl sulfotransferaseheterologously expressed in a microorganism exemplified by E. colisupplied with the substrate.

TABLE 2 Production of zosteric acid in E. coli from pHCA through theheterologous expression of sulfotransferases. pHCA Zosteric acid Enzymeremaining (mM) formed (mM) No enzyme 0.10 Not detectable SULT1A1 Rattusnorvegicus 0.02 0.10 SULT1A1 Homo sapiens 0.08 0.02 SULT1A1 Equuscaballus 0.09 0.01 SULT1A1 Sus scrofa domesticus 0.09 0.01 SULT1A1 Canislupus familiaris 0.10 0.01 SULT1E1 Gallus gallus domesticus 0.08 0.01

Example 2—Increased Production of Zosteric Acid in E. coli

The addition of sulfated groups to targets is dependent on supply of thedonor molecule 3′-Phosphoadenosine 5′-phosphosulfate (PAPS). We examinedif we could increase the production of zosteric acid by overexpressingenzymes providing PAPS and an enzyme that removes the product3′-Phosphoadenosine 5′-phosphate (PAP).

TABLE 3 Cloning of enzymes involved in activating sulfate and productremoval. Genes Fwd Primer Rev Primer cysDNC alone CBJP491 CBJP492 cysDNCfor artificial operon CBJP491 CBJP497 cysQ for artificial operon CBJP498CBJP496

In E. coli, the genes cysD and cysN encode the two subunits of ATPsulfurylase (EC:2.7.7.4), cysC encodes APS kinase (EC:2.7.1.25), andcysQ encode a PAP phosphatase.

The cysDNC cluster was amplified by PCR from E. coli MG1655 chromosomalDNA using the primers shown in table 3. The plasmid pRSFDuet-1 (LifeTechnologies) was digested by the restriction endonucleases NdeI andBglII. The gene cluster was inserted into the digested plasmid using theGibson reaction (New England Biolabs). FIG. 2 shows the resultingplasmid. For the combined expression of cysDNC and cysQ in an artificialoperon, cysDNCQ, the two parts were amplified by PCR from E. coli MG1655chromosomal DNA using the primers shown in Table 3. Again the parts wereinserted into the digested plasmid. FIG. 3 shows the resulting plasmids.The plasmid expressing SULT1A1 Homo sapiens (SEQ ID NO: 2) from example1 was co-transformed into E. coli BL21(DE3)pLysS cells (LifeTechnologies) with either the plasmid expressing cysDNC or cysDNCQ.

Cells were grown as in Example 1 and the supernatants were analyzed forproduct formation as in example 1. The strain expressing SULT1A1 incombination with cysDNCQ was also grown without the addition of IPTIGfor induction. Table 4 shows the concentrations of pHCA and zostericacid.

TABLE 4 Concentrations of pHCA and zosteric acid in culture media withE. coli expressing an aryl sulfotransferase in combination with cysDNCand cysQ. pHCA Zosteric acid remaining formed Enzymes Induction (mM)(mM) SULT1A1 Homo sapiens 0.1 mM IPTG 0.08 0.02 SULT1A1 Homo sapiens,0.1 mM IPTG 0.06 0.06 CysDNC SULT1A1 Homo sapiens, 0.1 mM IPTG 0.04 0.09CysDNCQ SULT1A1 Homo sapiens, None 0.10 Not detectable CysDNCQ

This shows that more of the pHCA is transformed into zosteric acid whenthe protein expression of cysDNC is increased. Even more zosteric acidis formed when the protein expression cysQ is additionally increased.

Example 3—A Sulfated Product can be Formed In Vivo by Co-Expression ofan Heterologous Pathway and an Aryl Sulfotransferase

The production of a sulfated product can be accomplished biologically bythe expression of aryl sulfotransferase as shown in example 1. Thesubstrate for sulfation may also be formed by a biological organism, andhere it will be shown for an organism expressing both a heterologouspathway leading to a phenolic compound and expressing a sulfotransferaseacting upon the phenolic compound.

The enzyme RmXAL from Rhodotorula mucilaginosa/Rhodotorula rubra (SEQ IDNO: 20) has tyrosine ammonia lyase activity, thus catalyzing thenon-oxidative deamination of the amino acid tyrosine, releasingp-coumaric acid (pHCA) and ammonia. The gene encoding RmXAL was codonoptimized using standard algorithms for expression in E. coli availableby GeneArt (Life Technologies) and amplified by PCR using the primersshown in table 5 and inserted into the pCDFDuet-1 vector (Novagen/LifeTechnologies), which had been digested by the restriction enzymes NdeIand Bglll, using Gibson reaction (New England Biolabs). FIG. 4 providesan image of the plasmid expressing RmXAL.

TABLE 5 Primers used for cloning of tyrosine ammonialyase Genes FwdPrimer Rev Primer RmXAL CBJP487 CBJP488

The resulting plasmid was co-transformed into E. coli BL21(DE3)pLysScells (Life Technologies) alone or together with the plasmid expressingSULT1A1 from Homo sapiens (example 1). The resulting strains was grownin M9 media with glucose as a carbon source, with 0.1 mM IPTG forinduction of gene expression. Samples were taken as described previously(example 1) for analysis of product formation. Table 6 shows theresulting concentrations of pHCA and zosteric acid. RmXAL allowed theproduction of pHCA without addition of any substrate, thus providing aheterologous pathway from the cells normal metabolism to a heterologousproduct. The additional expression of an aryl sulfotransferase,exemplified by SULT1A1 from Homo sapiens, allowed the in vivo conversionof pHCA to zosteric acid. Thus, an aryl sulfotransferase can act upon acompound produced in vivo and the cells can release the resultingsulfated product to the medium.

TABLE 6 Concentrations of pHCA and zosteric acid in culture media withE. coli expressing an aryl sulfotransferase in combination with atyrosine ammonia lyase. Enzymes pHCA (mM) Zosteric acid formed (mM)RmXAL 0.04 Not detectable SULT1A1 Homo sapiens, 0.02 0.01 RmXAL

Example 4—Decreased Toxicity of Sulfated Product

E. coli MG1655 was grown in chemically defined M9 minimal media with0.2% glucose as a carbon source without further addition or with theadditions of either 10 mM, 20 mM, 25 mM, 30 mM, 35 mM or 40 mMp-coumaric acid (pHCA), or with 20 mM or 40 mM of the sulfate ester ofpHCA (zosteric acid). All media preparations had been adjusted to pH 7.Cells were grown at 37° C. with 250 rpm shaking in an orbital shaker.The growth rates were examined by following the optical density at 600nm. The resulting growth rates in exponential growth phase are shown inFIG. 5. Filled squares represent growth rates in media with pHCA. Opensquares represent growth rates in media with zosteric acid. And thecircle represents the growth rate in media without any of theseadditions. It is evident that the presence of pHCA is toxic to thecells, while the sulfate ester, zosteric acid is much less so.

Example 5—In Vivo Supply of Precursor of Sulfated Product

The substrate that is the subject for sulfation may be supplied to themedium void of such precursors or may be provided by microorganisms inthe medium. Here we show that p-coumaric acid that is sulfated togenerate zosteric acid, can be produced in vivo by the expression of atyrosine ammonia-lyase.

The genes encoding the tyrosine ammonia-lyases RcTAL (from Rhodobactercapsulatus; SEQ ID NO: 48), RsTAL (from Rhodobacter sphaeroides; SEQ IDNO: 17) and FjTAL (from Flavobacterium johnsoniae; SEQ ID NO: 14) werecloned into expression vectors as follows. Genes (SEQ ID NO: 49, 50, and51, respectively) were optimized for E. coli and synthesized by GeneArt,amplified by PCR using the oligonucleotides shown in the table below,and cloned into pCDFDuet-1 (Novagen): The plasmid was digested with NdeIand BglII and gel purified. The genes were inserted by isothermalassembly using Gibson Assembly Master Mix (New England Biolabs), andtransformed into chemically competent DH5α (laboratory strain) or NEB5α(New England Biolabs), selecting for resistance to 50 μg mL⁻¹spectinomycin in LB medium. Resulting plasmids pCBJ215 (RsTAL), pCBJ228(FjTAL) and pCBJ297 (RcTAL) (FIGS. 6 to 8, respectively) wereco-transformed by electroporation into the E. coli expression strainBL21(DE3) (Invitrogen/Life Technologies) together with a pETDuet-1-basedplasmid expressing SULT1A1 from rat (Example 1). Transformation cultureswere plated on LB containing 50 μg mL⁻¹ spectinomycin and 100 μg mL⁻¹ampicillin. A control strain carrying pCDFDuet-1 was also made.

Primers:

Oligo- nucleotide Gene Direction Sequence CBJP483 RsTAL ForwardCATCTTAGTATATTAGTTAAGTATAAGAAGGAG ATATACATATGCTGGCAATGAGCCCT CBJP484RsTAL Reverse TGGCCGGCCGATATCCAATTGATTAAACCGGAC TCTGTTGC CBJP555 FjTALForward CATCTTAGTATATTAGTTAAGTATAAGAAGGAGATATACATATGAACACCATCAACGAATATCTG CBJP556 FjTAL ReverseTGGCCGGCCGATATCCAATTGATTAATTGTTAA TCAGGTGGTCTTTTACTTTCTG CBJP745 RcTALForward CATCTTAGTATATTAGTTAAGTATAAGAAGGAG ATATACATATGCTGGATGCAACCATTGGCBJP746 RcTAL Reverse TGGCCGGCCGATATCCAATTGATTATGCCGGA GGATCCGCT

Strains harboring recombinant plasmids were pre-cultured in 2xYT liquidmedium with 100 μg mL⁻¹ ampicillin and 50 μg mL⁻¹ spectinomycin andincubated at 37° C. and 250 rpm overnight. The following day, eachpre-culture was transferred into 5 ml of M9 minimal medium with 0.2%glucose, 2 mM tyrosine and 1 mM IPTG for induction of expression.Cultures were placed in an incubator at 37° C. with shaking at 250 rpmovernight. The supernatants were then collected by centrifugation twiceand applied to HPLC analysis as described in example 1, and the titersof p-coumaric acid (pHCA) and zosteric acid (ZA) were quantified usingchemical standards and are presented in the table below.

Sulfotransferase Tyrosine ammonia-lyase μM pHCA μM ZA SULT1A1 rat None 00 SULT1A1 rat RsTAL 78 <1 SULT1A1 rat RcTAL 20 <1 SULT1A1 rat FjTAL 39816

Here, it is evident that the zosteric acid is formed when there is asupply of exogenous p-coumaric acid or if the cells are capable ofproducing p-coumaric acid. Conclusively, a sulfated product may beformed from an unsulfated precursor molecule, when this is produced invivo.

Furthermore, the data surprisingly show that employing the tyrosineammonia-lyase from Flavobacterium johnsoniae (FjTAL; SEQ ID NO: 14)results in a higher supply in unsulfated precursor molecule (here:p-coumaric acid), which in turn leads to a higher yield of sulfatedproduct (here: zosteric acid) compare to other tyrosine ammonia-lyases.

Example 6—Production of Sulfated Products in Other Hosts

We have shown that zosteric acid can be produced in vivo in Escherichiacoli by expression of an aryl sulfotransferase. To show that thereaction is possible in other microorganisms, we here show that theyeast Saccharomyces cerevisiae can also be used as a host for theproduction.

The gene encoding aryl sulfotransferase SULT1A (Example 1) was clonedafter a TEF1 promoter into an episomal plasmid with a 2-micron origin ofreplication as follows. The gene was amplified by PCR using primersCBJP633 and CBJP634. Alternatively, the gene was codon-optimized for E.coli and synthesized by GeneArt and amplified by primers CBJP635 andCBJP636. The TEF1 promoter (Jensen et al., 2014, FEMS Yeast Res 14:238-248) was amplified by PCR using the primers PTEF1_fw and PTEF1_rv.Plasmid pCfB132 (Jensen et al., supra) was digested by restrictionenzymes AsiSI and Nt.BsmI. The three fragments—plasmid, TEF1 promotorand SULT1A1-encoding gene—were assembled using a uracil-excissioncloning procedure, resulting in plasmids pCBJ283 and pCBJ284 (FIGS. 9and 10, respectively, which was subsequently transformed into theSaccharomyces cerevisiae strain CEN.PK102-5B selecting for growth onsynthetic dropout media plates lacking uracil. A control strain was alsomade by transformation of pCfB132 into CEN.PK102-5B.

Primers:

Oligo- Gene/ nucleotide promoter Direction Sequence CBJP633 SULT1A1 ratForward AGTGCAGGUAAAACAATGgagttctcccgtcca CBJP634 SULT1A1 rat ReverseCGTGCGAUTCAtagttcacaacgaaacttg CBJP635 SULT1A1 rat ForwardATCTGTCAUAAAACAATGgaattttcacgtccgc (E. coli) CBJP636 SULT1A1 rat ReverseCACGCGAUTCAcagttcacaacgaaatttgaa (E. coli) PTEF1_fw PTEF1 ForwardCacgcgaugcacacaccatagcttc PTEF1_rv PTEF1 ReverseCgtgcgauggaagtaccttcaaaga

The strains were grown in modified Delft medium (Jensen et al., supra)with 20 mg/mL histidine and 60 mg/mL leucine and 10 mM p-coumaric acidovernight at 30° C. with aeration. The supernatant was then isolated andexamined by HPLC as described in Example 1. The table below shows thatzosteric acid (ZA) was produced by the strain expressing SULT1A1 and notthe control strain lacking a sulfotransferase.

μM ZA (averages and standard Sulfotransferase deviations of replicateexperiments) None  0 ± 0 SULT1A1 rat (native) 37.8 ± 5.7 SULT1A1 rat(codon 46.2 ± 3.5 optimized for E. coli)

It is evident that zosteric acid is formed only when a sulfotransferaseis expressed in yeast, and that the gene encoding this may be natural orencoded by a synthetic gene with a specific codon-optimization.Conclusively, the sulfation reactions shown to be catalyzed bysulfotransferases in E. coli are also catalyzed when thesulfotransferases are expressed in other organisms, as demonstrated herefor the yeast S. cerevisiae. The efficacy of production may be affectedby means such as the codon-usage of the genes encoding thesulfotransferase. Thus yeast expressing sulfotransferases may be able todetoxify aromatic compounds such as p-coumaric acid, and form sulfatedproducts such as zosteric acid.

Example 7—A Range of Compounds are Substrates for Sulfation In Vivo

Here we show that the expression of an aryl sulfotransferase may be ableto convert several substrates. Some of these are inhibitors that can befound in biomass hydrolyzate used as a substrate for cell growth andproduction in biotechnology. The compounds also include some that are ofbiotechnological interest as products of a cell culture or be some whosesulfate ester is of economic interest.

Different sulfotransferases were examined for their substratespecificities against three substrates. We tested the sulfotransferasesmentioned in example 1, as well as additional ones. The genes encodingthese were cloned as described in example 1 using the primers shown inthe table below from cDNA libraries of the respective organisms, exceptfor the SULT1A1 from rat (Rattus norvegicus) codon-optimized for E. coli(described above). The resulting vectors were transformed intoBL21(DE3)pLysS.

Primers:

Oligo- nucleotide Gene Direction Sequence CBJP517 SULT1C1 Gallus ForwardTAGAAATAATTTTGTTTAACTTTA gallus  AGAAGGAGATATACCatggccctgg domesticusataaaatgg CBJP518 SULT1C1 Gallus Reverse TAAGCATTATGCGGCCGCAAGCT gallusTGtcacaattccatgcgaaaaactag domesticus CBJP533 SULT1A1 Rattus ForwardTAGAAATAATTTTGTTTAACTTTA norvegicus AGAAGGAGATATACCatggaattttc(Codon-optimized acgtcc for E. coli) CBJP534 SULT1A1 Rattus ReverseTAAGCATTATGCGGCCGCAAGCT norvegicus TGttacagttcacaacgaaatttg(Codon-optimized for E. coli)

The resulting strains were grown in M9 medium containing either 100 μMpHCA, 95 μM resveratrol or 87 μM kaempferol. The cultures were grownovernight at 37° C., 300 rpm. The following day the supernatants wereisolated and examined by HPLC as described in example 1. BL21(DE3)pLysSwere used as a control strain and did not convert the substrates.

pHCA resveratrol kaempferol Enzyme 100 μM 95 μM 87 μM SULT1A1 Rattusnorvegicus 93% 93% 95% SULT1C1 Gallus gallus 26% 100% 80% domesticusSULT1A1 Rattus norvegicus 73% 58% 38% (Codon-optimized for E. coli)SULT1A1 human 39% 36% 97% SULT1A1 Equus caballus 21% 100% 96% SULT1E1Gallus gallus 17% 100% 47% domesticus SULT1A1 Canis lupus familiaris 34%61% 60% SULT1A1 Sus scrofa domesticus 8% 88% 45%

The table shows the percent conversion of the various substrates bycells expressing the different sulfotransferases. The results show thatseveral sulfotransferases, and especially the aryl sulfotransferase fromrat (Rattus norvegicus), may be employed in the sulfation of phenoliccompounds.

To further test the range of substrates that can be sulfated, we usedstrains carrying plasmids expressing SULT1A1 from rat (Rattusnorvegicus) and SULT1E1 from chicken (Gallus gallus domesticus)(Example 1) cloned into the expression vector pETDuet-1, and cysDNCQfrom E. coli cloned into expression vector pRSFDuet-1 (Example 2). Theplasmids were introduced into the E. coli expression strainBL21(DE3)pLysS as described previously, selecting for transformants withappropriate antibiotics, namely 34 μg mL⁻¹ chloramphenicol for pLysS,100 μg mL′ ampicillin for pETDuet-1-based vectors, and 100 μg mL⁻¹kanamycin for pRSFDuet-1-based vectors. The table below shows thecombination of over-expressed genes on plasmids. A control strainwithout a sulfotransferase gene or cysDNCQ operon was also examined.

E. coli strains Sulfotransferase Cys genes Control strain — — SULT1A1rat SULT1A1 rat — SULT1E1 chicken SULT1E1 chicken — SULT1A1 rat +CysDNCQ SULT1A1 rat CysDNCQ

The strains were precultured in 2xYT medium with appropriateantibiotics. 10 μL of these precultures were used to inoculate M9 mediawith 1 mM IPTG and none or a single substrate for sulfation. Afterovernight growth at 37° C., 300 rpm the supernatants were withdrawn andexamined by HPLC as described in Example 1. The compounds were detectedby UV absorbance. The table below shows the percent reduction inconcentration in the strains expressing sulfotransferases alone or incombination with cysDNCQ genes when compared to the control strain.

Start concen- tration SULT1A1 + Compound in μM SULT1A1 SULT1E1 CysDNCQFerulic acid 110 72% 67% 100% Quercetin 85 75% 74% 81% 4-hydroxybenzoicacid 287 5% 4% 6% 4-acetamidophenol 114 24% 10% 30% 3-Hydroxy-4- 132 51%24% 62% methoxycinnamic acid 4-Hydroxyphenylpyruvic 255 47% 100% 64%acid 3-(4- 241 3% 1% 7% Hydroxyphenyl)pro- pionic acid Vanillic acid 17333% 0% 39% Luteolin 61 27% 0% 37% Apigenin 77 41% 98% 99% fisetin 81 98%98% 100%

Conclusively, a wide range of phenolic compounds are substrates forsulfotransferases. In the shown examples, the conversion is enhanced bythe overexpression of cysDNCQ genes. Some of these compounds and theirsulfate esters are of interest in biotechnology. Also, some of thesecompounds are inhibitors of cell growth and function, and thusconversion by sulfation is of interest for use in biological systems.

Embodiments of the Invention

1. A process for the production of a sulfated phenolic compoundcomprising:

(i′) contacting a medium comprising a phenolic compound with a firstrecombinant host cell; wherein the first recombinant host cell comprisesa heterologous polypeptide having an aryl sulfotransferase activity; or

(i″) contacting a medium comprising a fermentable carbon substrate witha first recombinant host cell; wherein the first recombinant host cellcomprises a heterologous polypeptide having an aryl sulfotransferaseactivity; or

(i′″) contacting a medium comprising a precursor of a phenolic compoundwith a first recombinant host cell; wherein the first recombinant hostcell comprises a heterologous polypeptide having an arylsulfotransferase activity.

2. The process according to item 1, further comprising:

(ii) culturing the first recombinant host cell under suitable conditionsfor the production of the corresponding sulfated phenolic compound; and

(iii) optionally, recovering said sulfated phenolic compound.

3. The process according to item 1 or 2, wherein the heterologouspolypeptide having an aryl sulfotransferase activity is asulfotransferase 1A1 enzyme.

4. The process according to any one of items 1-3, wherein theheterologous polypeptide having an aryl sulfotransferase activity isselected from the group consisting of:

a) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1);

b) a polypeptide comprising an amino acid sequence which has at leastabout 70%, such as at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 93%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1); or

c) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1),wherein 1 to 50, such as 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1to 15, 1 to 10, 1 to 5, 1 to 3, amino acid residues are substituted,deleted and/or inserted.

5. The process according to any one of items 1-4, wherein theheterologous polypeptide is selected from the group consisting of:

a) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1;

b) a polypeptide comprising an amino acid sequence which has at leastabout 70%, such as at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 93%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 1; or

c) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, wherein 1 to 50, such as 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, amino acid residues aresubstituted, deleted and/or inserted.

6. The process according to any one of items 1-5, wherein the firstrecombinant host cell comprises an exogenous nucleic acid moleculecomprising a nucleotide sequence encoding said heterologous polypeptide.

7. The process according to item 6, wherein the exogenous nucleic acidmolecule further comprises a promoter that is functional in the hostcell to cause the production of an mRNA molecule and that is operablylinked to the nucleotide sequence encoding said heterologouspolypeptide.

8. The process according to item 6 or 7, wherein the exogenous nucleicacid molecule is a vector.

9. The process according to item 6 or 7, wherein the exogenous nucleicacid molecule is stabily integrated into the genome of said firstrecombinant host cell.

10. The process according to any one of items 1-7, wherein the firstrecombinant host cell has been further modified to have an increasedprotein expression of an ATP sulfurylase compared to an identical hostcell that does not carry said modification.

11. The process according to item 10, wherein the ATP sulfurylase isencoded by the genes cysD and cysN.

12. The process according to any one of items 1-11, wherein said firstrecombinant host cell has been further modified to have an increasedporetin expression of an APS kinase compared to an identical host cellthat does not carry said modification.

13. The process according to item 12, wherein the said APS kinase isencoded by the gene cysC.

14. The process according to any one of items 1-13, wherein said firstrecombinant host cell has been further modified to have an increasedprotein expression of a PAP phosphatase compared to an identical hostcell that does not carry said modification.

15. The process according to item 14, wherein said PAP phosphatase isencoded by the gene cycQ.

16. The process according to any one of items 10-15, wherein theincrease in protein expression is achieved by increasing the number ofcopies of the encoding gene or genes.

17. The process according to item 16, wherein the increase in the numberof copies of the gene or genes is achieved by using one or more vectorscomprising the gene or genes operably linked to a promoter that isfunctional in the host cell to cause the production of an mRNA molecule.

18. The process according to any one of items 10-15, wherein theincrease in protein expression is achieved by modifying the ribosomebinding site.

19. The process according to any one of items 10-18, wherein theincrease in protein expression is achieved by increasing the strength ofthe promoter(s) operably linked to the gene or genes.

20. The process according to any one of items 1-19, wherein said firstrecombinant host cell further comprises a heterologous polypeptidehaving a tyrosine ammonia lyase activity.

21. The process according to any one of items 1-20, wherein in step(i′), (i″) or (i′″) the medium is further contacted with a secondrecombinant host cell comprising a heterologous polypeptide having atyrosine ammonia lyase activity.

22. The process according to item 20 or 21, wherein the heterologouspolypeptide having a tyrosine ammonia lyase activity is selected fromthe group consisting of:

d) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 (e.g., SEQ ID NO: 14);

e) a polypeptide comprising an amino acid sequence which has at leastabout 70%, such as at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 93%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 14, 15, 16,17, 18, 19, 20, 21, 22 or 23 (e.g., SEQ ID NO: 14); or

f) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 (e.g., SEQ ID NO: 14),wherein 1 to 50, such as 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1to 15, 1 to 10, 1 to 5, 1 to 3, amino acid residues are substituted,deleted and/or inserted.

23. The process according to any one of items 20 to 22, wherein thefirst and/or second recombinant host cell comprises an exogenous nucleicacid molecule comprising a nucleotide sequence encoding saidheterologous polypeptide having a tyrosine ammonia lyase activity.

24. The process according to item 23, wherein the exogenous nucleic acidmolecule further comprises a promoter that is functional in the hostcell to cause the production of an mRNA molecule and that is operablylinked to the nucleotide sequence encoding said heterologouspolypeptide.

25. The process according to item 23 or 24, wherein the exogenousnucleic acid molecule is a vector.

26. The process according to item 23 or 24, wherein the exogenousnucleic acid is stably integrated into the genome of the first and/orsecond recombinant host cell.

27. The process according to any one of items 1 to 26, wherein the firstrecombinant host cell and the second recombinant host cell areindependently selected from the group consisting of bacteria, yeasts,fungi, algae and plant cells.

28. The process according to any one of items 1 to 27, wherein the firstrecombinant host cell is a bacterium.

29. The process according to item 28, wherein the bacterium is abacterium of the genus Bacillus, Lactococcus, Lactobacillus,Clostridium, Corynebacterium, Geobacillus, Thermoanaerobacterium,Streptococcus, Pseudomonas, Streptomyces, Escherichia, Shigella,Acinetobacter, Citrobacter, Salmonella, Klebsiella, Enterobacter,Erwinia, Kluyvera, Serratia, Cedecea, Morganella, Hafnia, Edwardsiella,Providencia, Proteus, or Yersinia.

30. The process according to item 28, wherein the bacterium is abacterium of the genus Bacillus.

31. The process according to item 30, wherein the bacterium is Bacillussubtilis.

32. The process according to item 28, wherein the bacterium is abacterium of the genus Lactococcus.

33. The process according to item 32, wherein the bacterium isLactococcus lactis.

34. The process according to item 28, wherein the bacterium is abacterium of the genus Pseudomonas.

35. The process according to item 34, wherein the bacterium isPseudomonas putida.

36. The process according to item 28, wherein the bacterium is abacterium of the genus Corynebacterium.

37. The process according to item 36, wherein the bacterium isCorynebacterium glutamicum.

38. The process according to item 28, wherein the bacterium is abacterium of the genus

Escherichia.

39. The process according to item 38, wherein the bacterium isEscherichia coli.

40. The process according to any one of item 1-27, wherein the firstrecombinant host cell is a yeast.

41. The process according to item 40, wherein the yeast is of the genusSaccharomyces, Pichia, Schizosacharomyces, Zygosaccharomyces, Hansenula,Pachyosolen, Kluyveromyces, Debaryomyces, Yarrowia, Candida,Cryptococcus, Komagataella, Lipomyces, Rhodospiridium, Rhodotorula, orTrichosporon.

42. The process according to item 40, wherein the yeast is a yeast ofthe genus Saccharomyces or Pichia.

43. The process according to item 40, wherein the yeast is selected fromthe group consisting of Saccharomyces cerevisiae, Pichia pastoris, andPichia kudriavzevii.

44. The process according to item 43, wherein the yeast is Saccharomycescerevisiae.

45. The process according to item 43, wherein the yeast is Pichiapastoris.

46. The process according to any one of items 1-27, wherein the firstrecombinant host cell is a fungus.

47. The process according to item 46, wherein the fungus is a fungus ofthe genus Aspergillus.

48. The process according to item 47, wherein the fungus is AspergillusOryzae or Aspergillus niger.

49. The process according to any one of items 1-27, wherein the firstrecombinant host cell is an algae cell.

50. The process according to item 49, wherein the algae cells is analgae cell of the genus Haematococcus, Phaedactylum, Volvox orDunaliella.

51. The process according to any one of items 1-27, wherein the firstrecombinant host cell is a plant cell.

52. The process according to item 51, wherein the plant cell is selectedfrom the group consisting of soybean, rapeseed, sunflower, cotton, corn,tobacco, alfalfa, wheat, barley, oats, sorghum, lettuce, rice, broccoli,cauliflower, cabbage, parsnips, melons, carrots, celery, parsley,tomatoes, potatoes, strawberries, peanuts, grapes, grass seed crops,sugar beets, sugar cane, beans, peas, rye, flax, hardwood trees,softwood trees, and forage grasses.

53. The process according to any one of items 1-52, wherein the phenoliccompound is represented by the general formula (I):

wherein at least one of R₁, R₂, R₃, R₄, and R₅ being an hydroxyl group(—OH);

wherein R₁, R₂, R₃, R₄, R₅ and R₆ are independently selected from thegroup consisting of halide, hydrogen, hydroxyl (—OH), —OR₇, —OCOR₇,—NR₇R₈, —COR₇, —COOR₇, —SR₇, —OSO₃R₇, —OCSR₇, —POR₇R₈, alkyl, alkenyl,alkynyl, aryl, and heteroaryl; wherein R₇, and R₈ are independentlyselected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl,aryl, and heteroaryl;

wherein R₁, R₂, R₃, R₄, R₅ and R₆, are optionally linked with a bridgemember Y_(n), thereby forming one or more rings, Y_(n) being a bond or aC₁₋₁₂ alkyl or an aryl, a carbocyclic, a heterocyclic or aheteroaromatic structure having 1-3 rings, 3-8 ring members in each and0 to 4 heteroatoms, or a heteroalkyl comprising 1 to 12 heteroatomsselected from the group consisting of N, O, S, S(O)₁₋₂ and carbonyl, andwherein n is an integer between 1 and 12.

54. A process according to any one of the items 1-52, wherein thephenolic compound is represented by the general formula (II):

wherein at least one of R₁, R₂, R₃, R₄, and R₅ being an hydroxyl group(—OH);

wherein R₁, R₂, R₃, R₄, R₅ and R₆ are independently selected from thegroup consisting of halide, hydrogen, hydroxyl (—OH), —OR₇, —OCOR₇,—NR₇R₈, —COR₇, —COOR₇, —SR₇, —OSO₃R₇, —OCSR₇, —POR₇R₈, alkyl, alkenyl,alkynyl, aryl, and heteroaryl; wherein R₇, and R₈ are independentlyselected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl,aryl, and heteroaryl;

wherein R₁, R₂, R₃, R₄, R₅ and R₆, are optionally linked with a bridgemember Y_(n), thereby forming one or more rings, Y_(n) being a bond or aC₁₋₁₂ alkyl or an aryl, a carbocyclic, a heterocyclic or aheteroaromatic structure having 1-3 rings, 3-8 ring members in each and0 to 4 heteroatoms, or a heteroalkyl comprising 1 to 12 heteroatomsselected from the group consisting of N, O, S, S(O)₁₋₂ and carbonyl, andwherein n is an integer between 1 and 12.

55. The process according to any one of items 1-53, wherein theprecursor of a phenolic compound in step (i′″) is a compound of thegeneral Formula (p-I):

wherein at least one of R₁, R₂, R₃, R₄, and R₅ being an hydroxyl group(—OH);

wherein R₁, R₂, R₃, R₄, R₅ and R₆ are independently selected from thegroup consisting of halide, hydrogen, hydroxyl (—OH), —OR₇, —OCOR₇,—NR₇R₈, —COR₇, —COOR₇, —SR₇, —OSO₃R₇, —OCSR₇, —POR₇R₈, alkyl, alkenyl,alkynyl, aryl, and heteroaryl; wherein R₇, and R₈ are independentlyselected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl,aryl, and heteroaryl;

wherein R₁, R₂, R₃, R₄, R₅ and R₆, are optionally linked with a bridgemember Y_(n), thereby forming one or more rings, Y_(n) being a bond or aC₁₋₁₂ alkyl or an aryl, a carbocyclic, a heterocyclic or aheteroaromatic structure having 1-3 rings, 3-8 ring members in each and0 to 4 heteroatoms, or a heteroalkyl comprising 1 to 12 heteroatomsselected from the group consisting of N, O, S, S(O)₁₋₂ and carbonyl, andwherein n is an integer between 1 and 12.

56. The process according to any one of items 53-55, wherein R₆ is—COOR₇, wherein R₇ is selected from the group consisting of hydrogen,alkyl, alkenyl, alkynyl, aryl, and heteroaryl.

57. The process according to item 56, wherein R₇ is hydrogen. 58. Theprocess according to any one of items 53-57, wherein R₃ is hydroxyl(—OH).

59. The process according to any one of items 53-58, wherein each of R₁,R₂, R₄ and R₅ is hydrogen.

60. The process according to any one of items 53-58, wherein R₄ ishydroxyl (—OH).

61. The process according to item 60, wherein each of R₁, R₂, and R₅ ishydrogen.

62. The process according to any one of items 53-55, wherein each of R₁,R₃ and R₅ is hydrogen, each of R₂ and R₄ is hydroxyl (—OH), and R₆ isp-hydroxyphenyl.

63. A recombinant host cell comprising a first heterologous polypeptidehaving aryl sulfotransferase activity, such as a polypeptide selectedfrom the group consisting of:

a) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1);

b) a polypeptide comprising an amino acid sequence which has at leastabout 70%, such as at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 93%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1); or

c) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1),wherein 1 to 50, such as 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1to 15, 1 to 10, 1 to 5, 1 to 3, amino acid residues are substituted,deleted and/or inserted.

64. The recombinant host cell according to item 63, wherein theheterologous polypeptide is selected from the group consisting of:

a) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1;

b) a polypeptide comprising an amino acid sequence which has at leastabout 70%, such as at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 93%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 1; or

c) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, wherein 1 to 50, such as 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, amino acid residues aresubstituted, deleted and/or inserted.

65. The recombinant host cell according to item 63 or 64, wherein thepolypeptide according to b) or c) has aryl sulfotransferase activity.

66. The recombinant host cells according to any one of items 63-65, thehost cell comprises an exogenous nucleic acid molecule comprising anucleotide sequence encoding said first heterologous polypeptide.

67. The recombinant host cell according to item 66, wherein theexogenous nucleic acid molecule further comprises a promoter that isfunctional in the host cell to cause the production of an mRNA moleculeand that is operably linked to the nucleotide sequence encoding saidfirst heterologous polypeptide.

68. The recombinant host cell according to item 67, wherein theexogenous nucleic acid molecule further comprises at least oneregulatory element selected from a 5′ untranslated region (5′UTR) and 3′untranslated region (3′ UTR).

69. The recombinant host cell according to any one of items 66-68,wherein the exogenous nucleic acid is a vector.

70. The recombinant host cell according to any one of items 66-68,wherein the excogenous nucleic acid is stably integrated into the genomeof the host cell.

71. The recombinant host cell according to any one of items 63-70,wherein the recombinant host cell has further been modified to have anincreased protein expression of an ATP sulfurylase compared to anidentical host cell that does not carry said modification.

72. The recombinant host cell according to item 71, wherein said ATPsulfurylase is encoded by the genes cysD and cysN.

73. The recombinant host cell according to any one of items 63-72,wherein the recombinant host cell has further been modified to have anincreased protein expression of an APS kinase compared to an identicalhost cell that does not carry said modification.

74. The recombinant host cell according to item 73, wherein said APSkinase is encoded by the gene cysC.

75. The recombinant host cell according to any one of items 63-74,wherein the recombinant host cell has further been modified to have anincreased protein expression of a PAP phosphatase compared to anidentical host cell that does not carry said modification.

76. The recombinant host cell according to item 75, wherein said PAPphosphatase is encoded by the gene cycQ.

77. The recombinant host cell according to any one of items 63-76,wherein the increase in gene expression has been achieved by anincreased number of copies of the gene or genes.

78. The precombinant host cell according to item 77, wherein theincrease in the number of copies of the gene or genes is achieved byusing one or more vectors comprising the gene or genes operably linkedto a promoter that is functional in the host cell to cause theproduction of an mRNA molecule.

79. The recombinant host cell according to any one of item 63-76,wherein the increase in protein expression is achieved by modifying theribosome binding site.

80. The recombinaint host cell according to any one of items 63-76,wherein the increase in gene expression has been achieved by increasingthe strength of the promoter(s) operably linked to the gene or genes.

81. The recombinant host cell according to any one of items 63-80,further comprising a second heterologous polypeptide having tyrosineammonia lyase activity, such as a polypeptide selected from the groupconsisting of:

d) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 (e.g., SEQ ID NO: 14);

e) a polypeptide comprising an amino acid sequence which has at leastabout 70%, such as at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 93%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 14, 15, 16,17, 18, 19, 20, 21, 22 or 23 (e.g., SEQ ID NO: 14); or

f) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 (e.g., SEQ ID NO: 14),wherein 1 to 50, such as 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1to 15, 1 to 10, 1 to 5, 1 to 3, amino acid residues are substituted,deleted and/or inserted.

82. The recombinant host cell according to item 81, wherein theheterologous polypeptide according to e) or f) has tyrosine ammonialyase activity.

83. The recombinant host cell according to item 81 or 82, wherein therecombinant host cell comprises an exogenous nucleic acid moleculecomprising a nucleotide sequence encoding said second heterologouspolypeptide.

84. The recombinant host cell according to item 83, wherein theexogenous nucleic acid molecule further comprises a promoter that isfunctional in the host cell to cause the production of an mRNA moleculeand that is operably linked to the nucleotide sequence encoding saidsecond heterologous polypeptide.

85. The recombinant host cell according to item 83 or 84, wherein theexogenous nucleic acid molecule is a vector.

86. The recombinant host cell according to item 83 or 84, wherein theexogenous nucleic acid is stably integrated into the genome of therecombinant host cell.

87. The recombinant host cell according to any one of items 63-86,wherein the recombinant host cell is selected from the group consistingof bacteria, yeasts, fungi, algae and plant cells.

88. The recombinant host cell according to any one of items 63-86,wherein the recombinant host cell is a bacterium.

89. The recombinant host cell according to item 88, wherein thebacterium is a bacterium of the genus Bacillus, Lactococcus,Lactobacillus, Clostridium, Corynebacterium, Geobacillus, Streptococcus,Pseudomonas, Streptomyces, Escherichia, Shigella, Acinetobacter,Citrobacter, Salmonella, Klebsiella, Enterobacter, Erwinia, Kluyvera,Serratia, Cedecea, Morganella, Hafnia, Edwardsiella, Providencia,Proteus, or Yersinia.

90. The recombinant host cell according to item 88, wherein thebacterium is a bacterium of the genus Bacillus.

91. The recombinant host cell according to item 90, wherein thebacterium is Bacillus subtilis.

92. The recombinant host cell according to item 88, wherein thebacterium is a bacterium of the genus Lactococcus.

93. The recombinant host cell according to item 92, wherein thebacterium is Lactococcus lactis.

94. The recombinant host cell according to item 88, wherein thebacterium is a bacterium of the genus Pseudomonas.

95. The recombinant host cell according to item 94, wherein thebacterium is Pseudomonas putida.

96. The recombinant host cell according to item 88, wherein thebacterium is a bacterium of the genus Corynebacterium.

97. The recombinant host cell according to item 96, wherein thebacterium is Corynebacterium glutamicum.

98. The recombinant host cell according to item 88, wherein thebacterium is a bacterium of the genus Escherichia.

99. The recombinant host cell according to item 98, wherein thebacterium is Escherichia coli.

100. The recombinant host cell according to any one of items 63-86,wherein the recombinant host cell is a yeast.

101. The recombinant host cell according to item 100, wherein the yeastis of the genus

Saccharomyces, Pichia, Schizosacharomyces, Zygosaccharomyces, Hansenula,Pachyosolen, Kluyveromyces, Debaryomyces, Yarrowia, Candida,Cryptococcus, Komagataella, Lipomyces, Rhodospiridium, Rhodotorula, orTrichosporon.

102. The recombinant host cell according to item 100, wherein the yeastis a yeast of the genus Saccharomyces or Pichia.

103. The recombinant host cell according to item 100, wherein the yeastis selected from the group consisting of Saccharomyces cerevisiae,Pichia pastoris, and Pichia kudriavzevii.

104. The recombinant host cell according to item 103, wherein the yeastis Saccharomyces cerevisiae.

105. The recombinant host cell according to item 103, wherein the yeastis Pichia pastoris.

106. The recombinant host cell according to any one of items 63-86,wherein the recombinant host cell is a fungus.

107. The recombinant host cell according to item 106, wherein the fungusis a fungus of the genus Aspergillus.

108. The recombinant host cell according to item 107, wherein the fungusis Aspergillus Oryzae or Aspergillus niger.

109. The recombinant host cell according to any one of items 63-86,wherein the recombinant host cell is an algae cell.

110. The recombinant host cell according to item 109, wherein the algaecells is an algae cell of the genus Haematococcus, Phaedactylum, Volvoxor Dunaliella.

111. The recombinant host cell according to any one of items 63-86,wherein the recombinant host cell is a plant cell.

112. The recombinant host cell according to item 111, wherein the plantcell is selected from the group consisting of soybean, rapeseed,sunflower, cotton, corn, tobacco, alfalfa, wheat, barley, oats, sorghum,lettuce, rice, broccoli, cauliflower, cabbage, parsnips, melons,carrots, celery, parsley, tomatoes, potatoes, strawberries, peanuts,grapes, grass seed crops, sugar beets, sugar cane, beans, peas, rye,flax, hardwood trees, softwood trees, and forage grasses.

113. The recombinant host cell according to any one of items 63-112,which is employed as first recombinant host cell in the processaccording to any one of items 1-62.

114. Use of a polypeptide in the sulfation of a phenolic compound, saidpolypeptide having aryl sulfotransferase activity, such as a polypeptideselected from the group consisting of:

a) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1);

b) a polypeptide comprising an amino acid sequence which has at leastabout 70%, such as at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 93%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1); or

c) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1),wherein 1 to 50, such as 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1to 15, 1 to 10, 1 to 5, 1 to 3, amino acid residues are substituted,deleted and/or inserted.

115. The use according to item 114, wherein the polypeptide is selectedfrom the group consisting of:

a) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1;

b) a polypeptide comprising an amino acid sequence which has at leastabout 70%, such as at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 93%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 1; or

c) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, wherein 1 to 50, such as 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, amino acid residues aresubstituted, deleted and/or inserted.

116. The use according to item 114 or 115, wherein the phenolic compoundis of the general formula (I):

wherein at least one of R₁, R₂, R₃, R₄, and R₅ being an hydroxyl group(—OH);

wherein R₁, R₂, R₃, R₄, R₅ and R₆ are independently selected from thegroup consisting of halide, hydrogen, hydroxyl (—OH), —OR₇, —OCOR₇,—NR₇R₈, —COR₇, —COOR₇, —SR₇, —OSO₃R₇, —OCSR₇, —POR₇R₈, alkyl, alkenyl,alkynyl, aryl, and heteroaryl; wherein R₇, and R₈ are independentlyselected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl,aryl, and heteroaryl;

wherein R₁, R₂, R₃, R₄, R₅ and R₆, are optionally linked with a bridgemember Y_(n), thereby forming one or more rings, Y_(n) being a bond or aC₁₋₁₂ alkyl or an aryl, a carbocyclic, a heterocyclic or aheteroaromatic structure having 1-3 rings, 3-8 ring members in each and0 to 4 heteroatoms, or a heteroalkyl comprising 1 to 12 heteroatomsselected from the group consisting of N, O, S, S(O)₁₋₂ and carbonyl, andwherein n is an integer between 1 and 12.

117. The use according to item 114 or 115, wherein the phenolic compoundis of the general formula (II)

wherein at least one of R₁, R₂, R₃, R₄, and R₅ being an hydroxyl group(—OH);

wherein R₁, R₂, R₃, R₄, R₅ and R₆ are independently selected from thegroup consisting of halide, hydrogen, hydroxyl (—OH), —OR₇, —OCOR₇,—NR₇R₈, —COR₇, —COOR₇, —SR₇, —OSO₃R₇, —OCSR₇, —POR₇R₈, alkyl, alkenyl,alkynyl, aryl, and heteroaryl; wherein R₇, and R₈ are independentlyselected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl,aryl, and heteroaryl;

wherein R₁, R₂, R₃, R₄, R₅ and R₆, are optionally linked with a bridgemember Y_(n), thereby forming one or more rings, Y_(n) being a bond or aC₁₋₁₂ alkyl or an aryl, a carbocyclic, a heterocyclic or aheteroaromatic structure having 1-3 rings, 3-8 ring members in each and0 to 4 heteroatoms, or a heteroalkyl comprising 1 to 12 heteroatomsselected from the group consisting of N, O, S, S(O)₁₋₂ and carbonyl, andwherein n is an integer between 1 and 12.

118. The use according to item 114 or 115, wherein the phenolic compoundis p-coumaric acid.

119. Process for the production of a sulfated phenolic compound, such aszosteric acid, the method comprises sulfating a phenolic compound, suchas p-coumaric acid, using a polypeptide having aryl sulfotransferaseactivity, such as a polypeptide selected from the group consisting of:

a) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1);

b) a polypeptide comprising an amino acid sequence which has at leastabout 70%, such as at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 93%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1); or

c) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1),wherein 1 to 50, such as 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1to 15, 1 to 10, 1 to 5, 1 to 3, amino acid residues are substituted,deleted and/or inserted.

120. The process according to item 119, wherein the polypeptide isselected from the group consisting of:

a) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1;

b) a polypeptide comprising an amino acid sequence which has at leastabout 70%, such as at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 93%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 1; or

c) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, wherein 1 to 50, such as 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, amino acid residues aresubstituted, deleted and/or inserted.

121. The process according to item 119 or 120, wherein the phenoliccompound is of the general formula (I) or (II) as defined herein.

122. A composition comprising a first recombinant host cell comprising aheterologous polypeptide having arylsulfotransferase activity, such as apolypeptide selected from the group consisting of:

a) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1);

b) a polypeptide comprising an amino acid sequence which has at leastabout 70%, such as at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 93%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1); or

c) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1),wherein 1 to 50, such as 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1to 15, 1 to 10, 1 to 5, 1 to 3, amino acid residues are substituted,deleted and/or inserted; and a second recombinant host cell comprising aheterologous polypeptide having tyrosine ammonia lyase activity, such asa polypeptide selected from the group consisting of:

d) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 (e.g., SEQ ID NO: 14);

e) a polypeptide comprising an amino acid sequence which has at leastabout 70%, such as at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 93%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 14, 15, 16,17, 18, 19, 20, 21, 22 or 23 (e.g., SEQ ID NO: 14); or

f) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 (e.g., SEQ ID NO: 14),wherein 1 to 50, such as 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1to 15, 1 to 10, 1 to 5, 1 to 3, amino acid residues are substituted,deleted and/or inserted.

123. A composition comprising a first polypeptide having arylsulfotransferase activity, such as a polypeptide selected from the groupconsisting of:

a) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1);

b) a polypeptide comprising an amino acid sequence which has at leastabout 70%, such as at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 93%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1); or

c) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., SEQ ID NO: 1),wherein 1 to 50, such as 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1to 15, 1 to 10, 1 to 5, 1 to 3, amino acid residues are substituted,deleted and/or inserted; and a second polypeptide having tyrosineammonia lyase activity, such as a polypeptide selected from the groupconsisting of:

d) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 (e.g., SEQ ID NO: 14);

e) a polypeptide comprising an amino acid sequence which has at leastabout 70%, such as at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 93%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 14, 15, 16,17, 18, 19, 20, 21, 22 or 23 (e.g., SEQ ID NO: 14); or

f) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 (e.g., SEQ ID NO: 14),wherein 1 to 50, such as 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1to 15, 1 to 10, 1 to 5, 1 to 3, amino acid residues are substituted,deleted and/or inserted.

1. A process for the production of a sulfated phenolic compoundcomprising: (i′) contacting a medium comprising a phenolic compound witha first recombinant host cell, wherein the first recombinant host cellcomprises a heterologous polypeptide having an aryl sulfotransferaseactivity; or (i″) contacting a medium comprising a fermentable carbonsubstrate with a first recombinant host cell; wherein the firstrecombinant host cell comprises a heterologous polypeptide having anaryl sulfotransferase activity; or (i′″) contacting a medium comprisinga precursor of a phenolic compound with a first recombinant host cell,wherein the first recombinant host cell comprises a heterologouspolypeptide having an aryl sulfotransferase activity. 2-38. (canceled)39. The process according to claim 1, further comprising: (ii) culturingthe first recombinant host cell under suitable conditions for theproduction of the corresponding sulfated phenolic compound; and (iii)optionally, recovering said sulfated phenolic compound.
 40. The processaccording to claim 1, wherein the heterologous polypeptide having anaryl sulfotransferase activity is a sulfotransferase 1A1 enzyme.
 41. Theprocess according to claim 1, wherein the heterologous polypeptidehaving an aryl sulfotransferase activity is selected from the groupconsisting of: a) a polypeptide comprising an amino acid sequence setforth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13; b) apolypeptide comprising an amino acid sequence which has at least about70% sequence identity to the amino acid sequence set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, wherein the polypeptide hasaryl sulfotransferase activity; and c) a polypeptide comprising an aminoacid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12 or 13, wherein 1 to 50 amino acid residues are substituted, deletedand/or inserted, wherein the polypeptide has aryl sulfotransferaseactivity.
 42. The process according to claim 1, wherein the heterologouspolypeptide is selected from the group consisting of: a) a polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 1; b) apolypeptide comprising an amino acid sequence which has at least about70% sequence identity to the amino acid sequence set forth in SEQ ID NO:1, wherein the polypeptide has aryl sulfotransferase activity; and c) apolypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1,wherein 1 to 50 amino acid residues are substituted, deleted and/orinserted, wherein the polypeptide has aryl sulfotransferase activity.43. The process according to claim 1, wherein the first recombinant hostcell has been further modified to have an increased expression of an ATPsulfurylase protein as compared to an identical host cell that does notcarry said modification.
 44. The process according to claim 43, whereinthe ATP sulfurylase is encoded by the genes cysD and cysN.
 45. Theprocess according to claim 1, wherein said first recombinant host cellhas been further modified to have an increased expression of an APSkinase protein as compared to an identical host cell that does not carrysaid modification.
 46. The process according to claim 45, wherein thesaid APS kinase is encoded by the gene cysC.
 47. The process accordingto claim 1, wherein said first recombinant host cell has been furthermodified to have an increased expression of a PAP phosphatase protein ascompared to an identical host cell that does not carry saidmodification.
 48. The process according to claim 47, wherein said PAPphosphatase is encoded by the gene cycQ.
 49. The process according toclaim 1, wherein said first recombinant host cell further comprises aheterologous polypeptide having a tyrosine ammonia lyase activity. 50.The process according to claim 1, wherein in step (i′), (i″) or (i′″)the medium is further contacted with a second recombinant host cellcomprising a heterologous polypeptide having a tyrosine ammonia lyaseactivity.
 51. The process according to claim 49, wherein theheterologous polypeptide having a tyrosine ammonia lyase activity isselected from the group consisting of: a) a polypeptide comprising anamino acid sequence set forth in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20,21, 22 or 23; b) a polypeptide comprising an amino acid sequence whichhas at least about 70% sequence identity to the amino acid sequence setforth in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23, whereinthe polypeptide has tyrosine ammonia lyase activity; and c) apolypeptide comprising an amino acid sequence set forth in SEQ ID NO:14, 15, 16, 17, 18, 19, 20, 21, 22 or 23, wherein 1 to 50 amino acidresidues are substituted, deleted and/or inserted, wherein thepolypeptide has tyrosine ammonia lyase activity.
 52. The processaccording to claim 49, wherein the heterologous polypeptide having atyrosine ammonia lyase activity is selected from the group consistingof: a) a polypeptide comprising an amino acid sequence set forth in SEQID NO: 14; b) a polypeptide comprising an amino acid sequence which hasat least about 70% sequence identity to the amino acid sequence setforth in SEQ ID NO: 14, wherein the polypeptide has tyrosine ammonialyase activity; and c) a polypeptide comprising an amino acid sequenceset forth in SEQ ID NO: 14, wherein 1 to 50 amino acid residues aresubstituted, deleted and/or inserted, wherein the polypeptide hastyrosine ammonia lyase activity.
 53. The process according to claim 1,wherein the phenolic compound is represented by the general formula (I):

wherein at least one of R₁, R₂, R₃, R₄, or R₅ is an hydroxyl group(—OH); wherein R₁, R₂, R₃, R₄, R₅ and R₆ are independently selected fromthe group consisting of: halide, hydrogen, hydroxyl (—OH), —OR₇, —OCOR₇,—NR₇R₈, —COR₇, —COOR₇, —SR₇, —OSO₃R₇, —OCSR₇, —POR₇R₈, alkyl, alkenyl,alkynyl, aryl, and heteroaryl, wherein R₇, and R₈ are independentlyselected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl,aryl, and heteroaryl; wherein R₁, R₂, R₃, R₄, R₅ and R₆, are optionallylinked with a bridge member Y_(n) thereby forming one or more rings,Y_(n) being a bond or a C₁₋₁₂ alkyl or an aryl, a carbocyclic, aheterocyclic or a heteroaromatic structure having 1-3 rings, 3-8 ringmembers in each and 0 to 4 heteroatoms, or a heteroalkyl comprising 1 to12 heteroatoms selected from the group consisting of N, O, S, S(O)₁₋₂and carbonyl, and wherein n is an integer between 1 and
 12. 54. Aprocess according to claim 1, wherein the phenolic compound isrepresented by the general formula (II):

wherein at least one of R₁, R₂, R₃, R₄, or R₅ is an hydroxyl group(—OH); wherein R₁, R₂, R₃, R₄, R₅ and R₆ are independently selected fromthe group consisting of halide, hydrogen, hydroxyl (—OH), —OR₇, —OCOR₇,—NR₇R₈, —COR₇, —COOR₇, —SR₇, —OSO₃R₇, —OCSR₇, —POR₇R₈, alkyl, alkenyl,alkynyl, aryl, and heteroaryl, wherein R₇, and R₈ are independentlyselected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl,aryl, and heteroaryl; wherein R₁, R₂, R₃, R₄, R₅ and R₆, are optionallylinked with a bridge member Y_(n), thereby forming one or more rings,Y_(n) being a bond or a C₁₋₁₂ alkyl or an aryl, a carbocyclic, aheterocyclic or a heteroaromatic structure having 1-3 rings, 3-8 ringmembers in each and 0 to 4 heteroatoms, or a heteroalkyl comprising 1 to12 heteroatoms selected from the group consisting of N, O, S, S(O)₁₋₂and carbonyl, and wherein n is an integer between 1 and
 12. 55. Theprocess according to claim 1, wherein the precursor of a phenoliccompound in step (i′″) is a compound of the general Formula (p-I):

wherein at least one of R₁, R₂, R₃, R₄, or R₅ is an hydroxyl group(—OH); wherein R₁, R₂, R₃, R₄, R₅ and R₆ are independently selected fromthe group consisting of halide, hydrogen, hydroxyl (—OH), —OR₇, —OCOR₇,—NR₇R₈, —COR₇, —COOR₇, —SR₇, —OSO₃R₇, —OCSR₇, —POR₇R₈, alkyl, alkenyl,alkynyl, aryl, and heteroaryl; wherein R₇, and R₈ are independentlyselected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl,aryl, and heteroaryl; wherein R₁, R₂, R₃, R₄, R₅ and R₆, are optionallylinked with a bridge member Y_(n), thereby forming one or more rings,Y_(n) being a bond or a C₁₋₁₂ alkyl or an aryl, a carbocyclic, aheterocyclic or a heteroaromatic structure having 1-3 rings, 3-8 ringmembers in each and 0 to 4 heteroatoms, or a heteroalkyl comprising 1 to12 heteroatoms selected from the group consisting of N, O, S, S(O)₁₋₂and carbonyl, and wherein n is an integer between 1 and
 12. 56. Theprocess according to claim 53, wherein R₆ is —COOR₇ and R₇ is hydrogen.57. The process according to claim 53, wherein R₃ is hydroxyl (—OH). 58.The process according to claim 53, wherein each of R₁, R₂, R₄ and R₅ ishydrogen, R₃ is hydroxyl (—OH) and R₆ is —COOH.
 59. A recombinant hostcell comprising a first heterologous polypeptide having arylsulfotransferase activity, such as a polypeptide selected from the groupconsisting of: a) a polypeptide comprising an amino acid sequence setforth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13; b) apolypeptide comprising an amino acid sequence which has at least about70% sequence identity to the amino acid sequence set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, wherein the polypeptide hasaryl sulfotransferase activity; and c) a polypeptide comprising an aminoacid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12 or 13, wherein 1 to 50 amino acid residues are substituted, deletedand/or inserted, wherein the polypeptide has aryl sulfotransferaseactivity.
 60. The recombinant host cell according to claim 59, whereinthe heterologous polypeptide is selected from the group consisting of:a) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1; b) a polypeptide comprising an amino acid sequence which has atleast about 70% sequence identity to the amino acid sequence set forthin SEQ ID NO: 1, wherein the polypeptide has aryl sulfotransferaseactivity; and c) a polypeptide comprising an amino acid sequence setforth in SEQ ID NO: 1, wherein 1 to 50 amino acid residues aresubstituted, deleted and/or inserted, wherein the polypeptide has arylsulfotransferase activity.
 61. The recombinant host cell according toclaim 59, wherein the recombinant host cell has further been modified tohave an increased protein expression of an ATP sulfurylase compared toan identical host cell that does not carry said modification.
 62. Therecombinant host cell according to claim 61, wherein said ATPsulfurylase is encoded by the genes cysD and cysN.
 63. The recombinanthost cell according to claim 59, wherein the recombinant host cell hasfurther been modified to have an increased expression of an APS kinaseprotein as compared to an identical host cell that does not carry saidmodification.
 64. The recombinant host cell according to claim 63,wherein said APS kinase is encoded by the gene cysC.
 65. The recombinanthost cell according to claim 59, wherein the recombinant host cell hasfurther been modified to have an increased expression of a PAPphosphatase protein as compared to an identical host cell that does notcarry said modification.
 66. The recombinant host cell according toclaim 65, wherein said PAP phosphatase is encoded by the gene cycQ. 67.The recombinant host cell according to claim 59, further comprising asecond heterologous polypeptide having tyrosine ammonia lyase activity,wherein said polypeptide is selected from the group consisting of: a) apolypeptide comprising an amino acid sequence set forth in SEQ ID NO:14, 15, 16, 17, 18, 19, 20, 21, 22 or 23; b) a polypeptide comprising anamino acid sequence which has at least about 70% sequence identity tothe amino acid sequence set forth in SEQ ID NO: 14, 15, 16, 17, 18, 19,20, 21, 22 or 23, wherein the polypeptide has tyrosine ammonia lyaseactivity; and c) a polypeptide comprising an amino acid sequence setforth in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23, wherein 1to 50 amino acid residues are substituted, deleted and/or inserted,wherein the polypeptide has tyrosine ammonia lyase activity.
 68. Therecombinant host cell according to claim 59, further comprising a secondheterologous polypeptide having tyrosine ammonia lyase activity, whereinthe heterologous polypeptide is selected from the group consisting of:a) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 14; b) a polypeptide comprising an amino acid sequence which has atleast about 70% sequence identity to the amino acid sequence set forthin SEQ ID NO: 14, wherein the polypeptide has tyrosine ammonia lyaseactivity; and c) a polypeptide comprising an amino acid sequence setforth in SEQ ID NO: 14, wherein 1 to 50 amino acid residues aresubstituted, deleted and/or inserted, wherein the polypeptide hastyrosine ammonia lyase activity.
 69. The recombinant host cell accordingto claim 59, which is employed as first recombinant host cell in theprocess according to claim
 1. 70. Process for the production of asulfated phenolic compound, such as zosteric acid, the method comprisessulfating a phenolic compound, such as p-coumaric acid, using apolypeptide having aryl sulfotransferase activity, such as a polypeptideselected from the group consisting of: a) a polypeptide comprising anamino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12 or 13; b) a polypeptide comprising an amino acid sequencewhich has at least about 70% sequence identity to the amino acidsequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12or 13, wherein the polypeptide has aryl sulfotransferase activity; andc) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, wherein 1 to 50 aminoacid residues are substituted, deleted and/or inserted, wherein thepolypeptide has aryl sulfotransferase activity.
 71. The processaccording to claim 70, wherein the polypeptide is selected from thegroup consisting of: a) a polypeptide comprising an amino acid sequenceset forth in SEQ ID NO: 1; b) a polypeptide comprising an amino acidsequence which has at least about 70% sequence identity to the aminoacid sequence set forth in SEQ ID NO: 1, wherein the polypeptide hasaryl sulfotransferase activity; and c) a polypeptide comprising an aminoacid sequence set forth in SEQ ID NO: 1, wherein 1 to 50 amino acidresidues are substituted, deleted and/or inserted, wherein thepolypeptide has aryl sulfotransferase activity.
 72. A compositioncomprising: a first recombinant host cell comprising a heterologouspolypeptide having arylsulfotransferase activity, such as a polypeptideselected from the group consisting of: a) a polypeptide comprising anamino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12 or 13; b) a polypeptide comprising an amino acid sequencewhich has at least about 70% sequence identity to the amino acidsequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12or 13, wherein the polypeptide has aryl sulfotransferase activity; andc) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, wherein 1 to 50 aminoacid residues are substituted, deleted and/or inserted, wherein thepolypeptide has aryl sulfotransferase activity; and a second recombinanthost cell comprising a heterologous polypeptide having tyrosine ammonialyase activity, such as a polypeptide selected from the group consistingof: a) a polypeptide comprising an amino acid sequence set forth in SEQID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23; b) a polypeptidecomprising an amino acid sequence which has at least about 70% sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 14, 15, 16,17, 18, 19, 20, 21, 22 or 23, wherein the polypeptide has tyrosineammonia lyase activity; and c) a polypeptide comprising an amino acidsequence set forth in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or23, wherein 1 to 50 amino acid residues are substituted, deleted and/orinserted, wherein the polypeptide has tyrosine ammonia lyase activity.73. The composition according to claim 72, comprising: a firstrecombinant host cell comprising a heterologous polypeptide havingarylsulfotransferase activity selected from the group consisting of: a)a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:1; b) a polypeptide comprising an amino acid sequence which has at leastabout 70% sequence identity to the amino acid sequence set forth in SEQID NO: 1, wherein the polypeptide has aryl sulfotransferase activity;and c) a polypeptide comprising an amino acid sequence set forth in SEQID NO: 1, wherein 1 to 50 amino acid residues are substituted, deletedand/or inserted, wherein the polypeptide has aryl sulfotransferaseactivity; and a second recombinant host cell comprising a heterologouspolypeptide having tyrosine ammonia lyase activity selected from thegroup consisting of: a) a polypeptide comprising an amino acid sequenceset forth in SEQ ID NO: 14; b) a polypeptide comprising an amino acidsequence which has at least about 70% sequence identity to the aminoacid sequence set forth in SEQ ID NO: 14, wherein the polypeptide hastyrosine ammonia lyase activity; and c) a polypeptide comprising anamino acid sequence set forth in SEQ ID NO: 14, wherein 1 to 50 aminoacid residues are substituted, deleted and/or inserted, wherein thepolypeptide has tyrosine ammonia lyase activity.
 74. A compositioncomprising: a first polypeptide having aryl sulfotransferase activity,such as a polypeptide selected from the group consisting of: a) apolypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13; b) a polypeptide comprising anamino acid sequence which has at least about 70% sequence identity tothe amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12 or 13, wherein the polypeptide has aryl sulfotransferaseactivity; and c) a polypeptide comprising an amino acid sequence setforth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, wherein1 to 50 amino acid residues are substituted, deleted and/or inserted,wherein the polypeptide has aryl sulfotransferase activity; and a secondpolypeptide having tyrosine ammonia lyase activity, such as apolypeptide selected from the group consisting of: a) a polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 14, 15, 16,17, 18, 19, 20, 21, 22 or 23; b) a polypeptide comprising an amino acidsequence which has at least about 70% sequence identity to the aminoacid sequence set forth in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22or 23, wherein the polypeptide has tyrosine ammonia lyase activity; andc) a polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23, wherein 1 to 50 amino acidresidues are substituted, deleted and/or inserted, wherein thepolypeptide has tyrosine ammonia lyase activity.
 75. The compositionaccording to claim 74, comprising: a first polypeptide having arylsulfotransferase activity selected from the group consisting of: a) apolypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1;b) a polypeptide comprising an amino acid sequence which has at leastabout 70% sequence identity to the amino acid sequence set forth in SEQID NO: 1, wherein the polypeptide has aryl sulfotransferase activity;and c) a polypeptide comprising an amino acid sequence set forth in SEQID NO: 1, wherein 1 to 50 amino acid residues are substituted, deletedand/or inserted, wherein the polypeptide has aryl sulfotransferaseactivity; and a second polypeptide having tyrosine ammonia lyaseactivity selected from the group consisting of: a) a polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 14; b) apolypeptide comprising an amino acid sequence which has at least about70% sequence identity to the amino acid sequence set forth in SEQ ID NO:14, wherein the polypeptide has tyrosine ammonia lyase activity; and c)a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:14, wherein 1 to 50 amino acid residues are substituted, deleted and/orinserted, wherein the polypeptide has tyrosine ammonia lyase activity.